Secondary literature sources for ALAD

The following references were automatically generated.

Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL Jr
A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics.
Arch Biochem Biophys. 2013; 530: 73-82
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The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a approximately 90 degrees rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the intersubunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic.

Jaffe EK, Lawrence SH
The morpheein model of allostery: evaluating proteins as potential morpheeins.
Methods Mol Biol. 2012; 796: 217-31
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An equilibrium mixture of alternate quaternary structure assemblies can form a basis for allostery. The morpheein model of allostery is a concerted dissociative model that describes an equilibrium of alternate quaternary structure assemblies whose architectures are dictated by alternate conformations in the dissociated state. Kinetic and biophysical anomalies that suggest that the morpheein model of allostery applies for a given protein of interest are briefly described. Two methods are presented for evaluating proteins as potential morpheeins. One is a subunit interchange method that uses chromatography, dialysis, and mass spectroscopy to monitor changes in multimer composition. The other is a two-dimensional native gel electrophoresis method to monitor ligand-induced changes in an equilibrium of alternate multimeric assemblies.

Jaffe EK, Lawrence SH
Allostery and the dynamic oligomerization of porphobilinogen synthase.
Arch Biochem Biophys. 2012; 519: 144-53
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The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer <--> 2mer <--> 2mer *<--> 6mer *. The " *" represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH with the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer * relative to wild type. The 6mer * has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer *. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects.

Erdtman E, Bushnell EA, Gauld JW, Eriksson LA
Computational insights into the mechanism of porphobilinogen synthase.
J Phys Chem B. 2010; 114: 16860-70
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Porphobilinogen synthase (PBGS) is a key enzyme in heme biosynthesis that catalyzes the formation of porphobilinogen (PBG) from two 5-aminolevulinic acid (5-ALA) molecules via formation of intersubstrate C-N and C-C bonds. The active site consists of several invariant residues, including two lysyl residues (Lys210 and Lys263; yeast numbering) that bind the two substrate moieties as Schiff bases. Based on experimental studies, various reaction mechanisms have been proposed for this enzyme that generally can be classified according to whether the intersubstrate C-C or C-N bond is formed first. However, the detailed catalytic mechanism of PBGS remains unclear. In the present study, we have employed density functional theory methods in combination with chemical models of the two key lysyl residues and two substrate moieties in order to investigate various proposed reaction steps and gain insight into the mechanism of PBGS. Importantly, it is found that mechanisms in which the intersubstrate C-N bond is formed first have a rate-limiting barrier (17.5 kcal/mol) that is lower than those in which the intersubstrate C-C bond is formed first (22.8 kcal/mol).

Kade IJ et al.
Studies on the antioxidant effect and interaction of diphenyl diselenide and dicholesteroyl diselenide with hepatic delta-aminolevulinic acid dehydratase and isoforms of lactate dehydrogenase.
Toxicol In Vitro. 2009; 23: 14-20
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Studies on the interaction of dicholesteroyl diselenide (DCDS) and diphenyl diselenide (DPDS) with hepatic delta-aminolevulinic acid dehydratase (ALA-D) and different isoforms of lactate dehydrogenase (LDH) from different tissues were investigated. In addition, their antioxidant effects were tested in vitro by measuring the ability of the compounds to inhibit the formation of hepatic thiobarbituric acid reactive species (TBARS) induced by both iron (II) and sodium nitroprusside (SNP). The results show that while DPDS markedly inhibited the formation of TBARS induced by both iron (II) and SNP, DCDS did not. Also, the activities of hepatic delta-aminolevulinic acid dehydratase (ALA-D) and different isoforms of lactate dehydrogenase (LDH) were significantly inhibited by both DPDS and DCDS. Moreover, we further observed that the in vitro inhibition of different isoforms of lactate dehydrogenase by DCDS and DPDS likely involves the modification of the groups at the NAD+ binding site of the enzyme. Since organoselenides interacts with thiol groups on proteins, we conclude that the inhibition of different isoforms of lactate dehydrogenase by DPDS and DCDS possibly involves the modification of the thiol groups at the NAD+ binding site of the enzyme.

Ulbrichova D, Hrdinka M, Saudek V, Martasek P
Acute intermittent porphyria--impact of mutations found in the hydroxymethylbilane synthase gene on biochemical and enzymatic protein properties.
FEBS J. 2009; 276: 2106-15
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Acute intermittent porphyria is an autosomal dominantly inherited disorder, classified as acute hepatic porphyria, caused by a deficiency of hydroxymethylbilane synthase (EC 2.5.1.61, EC 4.3.1.8, also known as porphobilinogen deaminase, uroporphyrinogen I synthase), the third enzyme in heme biosynthesis. Clinical features include autonomous, central, motor or sensory symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. A diagnosis of acute intermittent porphyria is crucial to prevent life-threatening acute attacks. Detection of DNA variations by molecular techniques allows a diagnosis of acute intermittent porphyria in situations where the measurement of porphyrins and precursors in urine and faeces and erythrocyte hydroxymethylbilane synthase activity is inconclusive. In the present study, we identified gene defects in six Czech patients with acute intermittent porphyria, as diagnosed based on biochemical findings, and members of their families to confirm the diagnosis at the molecular level and/or to provide genetic counselling. Molecular analyses of the hydroxymethylbilane synthase gene revealed seven mutations. Four were previously reported: c.76C>T, c.77G>A, c.518G>A, c.771 + 1G>T (p.Arg26Cys, p.Arg26His, p.Arg173Gln). Three were novel mutations: c.610C>A, c.675delA, c.750A>T (p.Gln204Lys, p.Ala226ProfsX28, p.Glu250Asp). Of particular interest, one patient had two mutations (c.518G>A; c.610C>A), both located in exon 10 of the same allele. To establish the effects of the mutations on enzyme function, biochemical characterization of the expressed normal recombinant and mutated proteins was performed. Prokaryotic expression of the mutant alleles of the hydroxymethylbilane synthase gene revealed that, with the exception of the p.Gln204Lys mutation, all mutations resulted in little, if any, enzymatic activity. Moreover, the 3D structure of the Escherichia coli and human protein was used to interpret structure-function relationships for the mutations in the human isoform.

Saikeur A, Choorit W, Prasertsan P, Kantachote D, Sasaki K
Influence of precursors and inhibitor on the production of extracellular 5-aminolevulinic acid and biomass by Rhodopseudomonas palustris KG31.
Biosci Biotechnol Biochem. 2009; 73: 987-92
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5-Aminolevulinic acid (ALA) and the biomass of photosynthetic bacteria, Rhodopseudomonas palustris KG31, have very high potential for development and exploitation as bioherbicide and biofertilizer respectively. In this work, the effects of two precursors and an inhibitor of aminolevulinic dehydratase (ALAD) added to the VFA culture medium on the production of ALA and biomass were investigated. The experimental runs were carried out according to a Box-Behnken design. The precursors were added to the medium at the beginning of cultivation, while the inhibitor was added after 24 h. Statistical analysis indicated that levulinic acid (LA) has a positive effect on ALA production while glycine has a negative effect on biomass production. In order to enhance both ALA and biomass products, the most suitable medium was VFA medium supplemented with 3.0 mM glycine and 10 mM LA, giving ALA and biomass of 182.91 microM and 3.1 gDCW/l within 54 h.

Lin J, Fu W, Cen P
Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production.
Bioresour Technol. 2009; 100: 2293-7
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The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20mM D-glucose or D-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3g/l (56 mM) under the appropriate conditions in the fermenter.

Sardh E, Harper P, Andersson DE, Floderus Y
Plasma porphobilinogen as a sensitive biomarker to monitor the clinical and therapeutic course of acute intermittent porphyria attacks.
Eur J Intern Med. 2009; 20: 201-7
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BACKGROUND: Acute intermittent porphyria (AIP) is a metabolic disease affecting hepatic heme biosynthesis. The clinical course in overt disease is characterized by acute attacks of neurovisceral symptoms. Treatment is based on symptomatic relief together with carbohydrate loading and in more severe attacks heme therapy. During an acute attack the heme precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA) are produced in high amounts by the liver and are found in high concentrations in plasma and urine. These metabolites represent the acute phase reactants confirming an ongoing attack and are used to evaluate therapeutic measures. The aim of this study was to measure PBG and ALA in plasma and urine during an acute attack and to match the biochemical pattern with the clinical and therapeutical course. METHODS: Three consecutive AIP patients were included during four acute attacks. Plasma PBG and ALA were measured by a LC-MS method and in urine by ion-exchange chromatography. The patients received symptomatic and glucose treatment at admission to hospital, and four days later, if necessary, heme therapy. RESULTS: In the three attacks that required heme therapy, plasma PBG concentrations had further increased after admission (p=0.01). In the patient that did not require heme therapy, plasma PBG had decreased after admission. CONCLUSIONS: Biochemical monitoring of an acute attack was more accurately reflected by plasma PBG than plasma ALA or urinary PBG and ALA. Glucose administration, in contrast to heme therapy, was not sufficient to achieve clinical and biochemical remission in the more serious attacks.

Mogi T et al.
Siccanin rediscovered as a species-selective succinate dehydrogenase inhibitor.
J Biochem. 2009; 146: 383-7
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To identify antibiotics targeting to respiratory enzymes, we carried out matrix screening of a structurally varied natural compound library with Pseudomonas aeruginosa membrane-bound respiratory enzymes. We identified a succinate dehydrogenase inhibitor, siccanin (IC(50), 0.9 microM), which is a potent antibiotic against some pathogenic fungi like Trichophyton mentagrophytes and inhibits their mitochondrial succinate dehydrogenase. We found that siccanin was effective against enzymes from P. aeruginosa, P. putida, rat and mouse mitochondria but ineffective or less effective against Escherichia coli, Corynebacterium glutamicum, and porcine mitochondria enzyme. Action mode was mixed-type for quinone-dependent activity and noncompetitive for succinate-dependent activity, indicating the proximity of the inhibitor-binding site to the quinone-binding site. Species-selective inhibition by siccanin is unique among succinate dehydrogenase inhibitors, and thus siccanin is a potential lead compound for new chemotherapeutics.

Lawrence SH, Ramirez UD, Selwood T, Stith L, Jaffe EK
Allosteric inhibition of human porphobilinogen synthase.
J Biol Chem. 2009; 284: 35807-17
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Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

Luo Q, Olucha J, Lamb AL
Structure-function analyses of isochorismate-pyruvate lyase from Pseudomonas aeruginosa suggest differing catalytic mechanisms for the two pericyclic reactions of this bifunctional enzyme.
Biochemistry. 2009; 48: 5239-45
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The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions in a single active site. PchB physiologically produces salicylate and pyruvate from isochorismate for ultimate incorporation of the salicylate into the siderophore pyochelin. PchB also produces prephenate from chorismate, most likely due to structural homology to the Escherchia coli chorismate mutase. The molecular basis of catalysis among enzymatic pericyclic reactions is a matter of debate, one view holding that catalysis may be derived from electrostatic transition state stabilization and the opposing view that catalysis is derived from the generation of a reactive substrate conformation. Mutant forms of PchB were generated by site-directed mutagenesis at the site (K42) hypothesized to be key for electrostatic transition state stabilization (K42A, K42Q, K42E, and K42H). The loop containing K42 is mobile, and a mutant to slow loop dynamics was also designed (A43P). Finally, a previously characterized mutation (I87T) was also produced. Circular dichroism was used to assess the overall effect on secondary structure as a result of the mutations, and X-ray crystallographic structures are reported for K42A with salicylate and pyruvate bound and for apo-I87T. The data illustrate that the active site architecture is maintained in K42A-PchB, which indicates that differences in activity are not caused by secondary structural changes or by differences in active site loop conformation but rather by the chemical nature of this key residue. In contrast, the I87T structure demonstrates considerable mobility, suggesting that loop dynamics and conformational plasticity may be important for efficient catalysis. Finally, the mutational effects on k(cat) provide evidence that the two activities of PchB are not covariant and that a single hypothesis may not provide a sufficient explanation for catalysis.

Lu H, Tonge PJ
Inhibitors of FabI, an enzyme drug target in the bacterial fatty acid biosynthesis pathway.
Acc Chem Res. 2008; 41: 11-20
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The modern age of drug discovery, which had been slowly gathering momentum during the early part of the twentieth century, exploded into life in the 1940s with the isolation of penicillin and streptomycin. The immense success of these early drug discovery efforts prompted the general view that many infectious diseases would now be effectively controlled and even eradicated. However this initial optimism was misplaced, and pathogens such as multidrug-resistant Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus present a major current threat to human health. Drug resistance arises through the unrelenting pressure of natural selection, and there is thus a continuing need to identify novel drug targets and develop chemotherapeutics that circumvent existing drug resistance mechanisms. In this Account, we summarize current progress in developing inhibitors of FabI, the NADH-dependent enoyl reductase from the type II bacterial fatty acid biosynthesis pathway (FAS-II), a validated but currently underexploited target for drug discovery. The FabI inhibitors have been divided into two groups, based on whether they form a covalent adduct with the NAD (+) cofactor. Inhibitors that form a covalent adduct include the diazaborines, as well as the front-line tuberculosis drug isoniazid. The NAD adducts formed with these compounds are formally bisubstrate enzyme inhibitors, and we summarize progress in developing novel leads based on these pharmacophores. Inhibitors that do not form covalent adducts form a much larger group, although generally these compounds also require the cofactor to be bound to the enzyme. Using structure-based approaches, we have developed a series of alkyl diphenyl ethers that are nanomolar inhibitors of InhA, the FabI from M. tuberculosis, and that are active against INH-resistant strains of M. tuberculosis. This rational approach to inhibitor development is based on the proposal that high-affinity inhibition of the FabI enzymes is coupled to the ordering of a loop of amino acids close to the active site. Compounds that promote loop ordering are slow onset FabI inhibitors with increased residence time on the enzyme. The diphenyl ether skeleton has also been used as a framework by us and others to develop potent inhibitors of the FabI enzymes from other pathogens such as Escherichia coli, S. aureus, and Plasmodium falciparum. Meanwhile chemical optimization of compounds identified in high-throughput screening programs has resulted in the identification of several classes of heteroaromatic FabI inhibitors with potent activity both in vitro and in vivo. Finally, screening of natural product libraries may provide useful chemical entities for the development of novel agents with low toxicity. While the discovery that not all pathogens contain FabI homologues has led to reduced industrial interest in FabI as a broad spectrum target, there is substantial optimism that FabI inhibitors can be developed for disease-specific applications. In addition, the availability of genome sequencing data, improved methods for target identification and validation, and the development of novel approaches for determining the mode of action of current drugs will all play critical roles in the road ahead and in exploiting other components of the FAS-II pathway.

Zoossmann-Diskin A, Swinburne S, Shohat M, Peleg L, Gazit E, Turner D
Typing classical polymorphisms by real-time PCR: analysis of the GPT and ALAD protein polymorphisms in the Jewish populations.
Am J Hum Biol. 2008; 20: 490-2
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Expanding the already extensive data array on classical polymorphisms in new populations is beneficial to full characterization of the intricate relationships between human populations. This can be done in a short time on a large-scale by using real-time PCR. The red-cell enzymes Delta-aminolevulinate dehydratase (ALAD) and glutamate pyruvate transaminase (GPT) have each two alleles that can be separated by protein electrophoresis. They were previously characterized by protein electrophoresis in some of the Jewish populations. We designed primers and probes for fluorescence resonance energy transfer detection of the ALAD and GPT alleles to type Jewish populations including some in which these polymorphisms have not been studied in the past. The methods described here allow fast and efficient determination of these common classical polymorphisms. When used in large-scale population studies, these methods are not only faster, but also cheaper than amplification followed by restriction enzyme digestion and gel electrophoresis. The frequency of ALAD*1 among Jews ranges from 0.781 to 0.960 and that of GPT*1 from 0.412 to 0.773. A maximum likelihood tree based on the ALAD and GPT polymorphisms and five other red-cells enzymes polymorphisms depicts quite accurately the known historic, anthropological, and geographic relations between the Jewish populations.

Kokona B, Rigotti DJ, Wasson AS, Lawrence SH, Jaffe EK, Fairman R
Probing the oligomeric assemblies of pea porphobilinogen synthase by analytical ultracentrifugation.
Biochemistry. 2008; 47: 10649-56
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The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric assemblies, and under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild-type enzyme at elevated pH [Breinig, S., et al. (2003) Nat. Struct. Biol. 10, 757-763]. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized, and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well documented under some conditions, both assemblies are very stable (under AU conditions) in the time frame of our ultracentrifuge experiments.

Kadam RU, Garg D, Roy N
Selective mapping of chemical space for Pseudomonas aeruginosa deacetylase LpxC inhibitory potential.
Chem Biol Drug Des. 2008; 71: 45-56
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UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase enzyme of Pseudomonas aeruginosa is an interesting target for development of anti-infective drugs against this gram-negative bacterium. Many segregated studies analyzing the P. aeruginosa UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase and its inhibitors have been reported in the recent past. In the present study, an attempt has been made to integrate this knowledge for the development of an effective multilayer screening approach. Eventually, an extensive chemical space was screened to filter out three potential P. aeruginosa UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase inhibitors.

Shaik AP, Jamil K
A study on the ALAD gene polymorphisms associated with lead exposure.
Toxicol Ind Health. 2008; 24: 501-6
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Delta -aminolevulinic acid dehydratase (ALAD) plays an important role in lead poisoning, and polymorphisms in this gene may affect the response of individuals to lead toxicity symptoms. This study was carried out to examine the effects of ALAD gene polymorphism (G177C) on blood lead levels (BLL) and hematological parameters. In all, 113 battery manufacturing unit workers and 102 occupationally unexposed controls from Hyderabad, A.P, India formed the study group. Genotypes for the ALAD G177C polymorphism were determined by Polymerase chain reaction and restriction fragment length digestion. BLL were determined by anode stripping voltammetry using ESA Model 3010B Lead analyzer. Complete blood picture was analyzed using ADVIA cell counter for each sample. The frequencies for ALAD1 and ALAD2 alleles were 0.98 and 0.01, respectively. ALAD 1-2 and ALAD 2-2 genotypes together were considered as a single group and compared with the ALAD 1-1 genotype group. BLL did not differ significantly among ALAD1-1, 1-2, and 2-2 genotypes; however, subjects from the ALAD 1-2/2-2 genotype group showed higher BLL concentrations of 80.51 microg/dL when compared with subjects from the ALAD 1-1 genotype group (50.4 microg/dL). Approximately 29.2% volunteers (nA =A 33) from the occupationally exposed group had hemoglobin levels below 10.0 g/dL. There was no significant difference in total white cell count and platelet count between occupational and non-exposed lead-exposed groups. BLL of occupationally exposed individuals were significantly high compared with the unexposed group. ALAD G177C polymorphism along with BLL and assessment of hematological parameters may play an important role in evaluation and better understanding of the consequences of lead exposure.

Lee HJ, Chang HY, Venkatesan N, Peng HL
Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1.
FEBS Lett. 2008; 582: 3479-83
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Phosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co2+-dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8-20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI.

Steward RE, Armen RS, Daggett V
Different disease-causing mutations in transthyretin trigger the same conformational conversion.
Protein Eng Des Sel. 2008; 21: 187-95
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Transthyretin (TTR)-containing amyloid fibrils are deposited in cardiac tissue as a natural consequence of aging. A large number of inherited mutations lead to amyloid diseases by accelerating TTR deposition in other organs. Amyloid formation is preceded by a disruption of the quaternary structure of TTR and conformational changes in the monomer. To study conformational changes preceding the formation of amyloid, we performed molecular dynamics simulations of the wild-type monomer, amyloidogenic variants (V30M, L55P, V122I) and a protective variant (T119M) at neutral and low pH. At low pH, the D strand dissociated from the beta-sheet to expose the A strand, consistent with experimental studies. In amyloidogenic variants and in the wild-type at low pH, there was a conformational change in the beta-sheets into alpha-sheet via peptide bond flips that was not observed at neutral pH in the wild-type monomer. The same residues participated in conversion in each amyloidogenic variant simulation, originating in the G strand between residues 106 and 109, with accelerated conversion at low pH. The T119M protective variant changed the local conformation of the H strand and suppressed the conversion observed in amyloidogenic variants.

Cigerci IH, Korcan SE, Konuk M, Ozturk S
Comparison of ALAD activities of Citrobacter and Pseudomonas strains and their usage as biomarker for Pb contamination.
Environ Monit Assess. 2008; 139: 41-8
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Delta-aminolevulinate dehydratase (ALAD) activity has been used in prokaryotes and eukaryotes as a biomarker for environmental lead (Pb) exposure and toxicity. Microorganisms are sensitive indicators of toxicity at the fundamental level of ecological organization, but bacterial biomarker studies are focused on the Pseudomonas strains in Group I and E coli. The objectives of the present work were to determine if Burkholderia gladioli belonging to group II, due to its 16SrRNA similarity, can be used as biomarker in metal contamination and compare its possible usage with Pseudomonas aeruginosa and Citrobacter freundii (previously known as Esherichia freundii) and Bacterium freundii which are classified in Group I. In this study, ALAD activity in an environmental strains of Burkholderia gladioli, Pseudomonas aeruginosa, Citrobacter freundii were investigated to evaluate potential inhibition by Pb and other toxic metals. When the ALAD activity of Burkholderia gladioli was tested, Co and Pb decreased activity by 27 and 71%, respectively. In addition to these findings, Zn increased the activity up to 26%. These effects were found to be statistically meaningful (p < 0.05). It was determined that the increase of lead concentration inhibites the ALAD activity at each of the three strains. There was a statistically significant dose-response relationship between ALAD activity in cells of Burkholderia gladioli and Pb (Pearson correlation coefficent = -0.665; r(2) = 0.665, and p < 0.001). The strongest ALAD inhibition which was measured was 90% at Burkholderia gladioli when protein extracts were incubated with 750 muM of Pb. The relationship between Pb and ALAD activity was statistically described by [ALAD Activity] = 0.476-0.000597 x [Pb]. According to the obtained results, we suggest that the ALAD of Burkholderia gladioli can be used as a biomarker for lead contamination in the environment.

Montenegro MF, Barbosa F Jr, Tanus-Santos JE
Assessment of how pregnancy modifies plasma lead and plasma/whole blood lead ratio in ALAD 1-1 genotype women.
Basic Clin Pharmacol Toxicol. 2008; 102: 347-51
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Pregnant women are one of the most sensitive populations to the toxic effects associated with lead (Pb) exposure. These effects are primarily associated with plasma Pb (Pb-P), which reflects the most rapidly exchangeable fraction of Pb in the bloodstream, and elevated maternal Pb-P may be more relevant to foetal Pb exposure than whole blood Pb (Pb-B). We investigated how pregnancy affects Pb-B, Pb-P and %Pb-P/Pb-B ratios without the influence of the delta-aminolevulinic acid dehydratase (ALAD) G177C polymorphism, which is a major genetic factor influencing Pb-B, Pb-P and %Pb-P/Pb-B ratios. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion in nine pregnant and 20 non-pregnant women, aged 18-33, environmentally exposed to Pb. Here, we included only women with ALAD 1-1 genotype. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. We found no differences in Pb-B (P > 0.05). However, pregnant women had a 2-fold increase in Pb-P and a 3-fold increase in %Pb-P/Pb-B (both P < 0.01) compared to non-pregnant women. These alterations in Pb concentrations associated with pregnancy are similar to those associated with different ALAD gene variants. We can now better appreciate how pregnancy affects foetal exposure to Pb without the influence of this important genetic factor.

Raigal Martin MY, Lledo Navarro JL, Raigal Martin JM, Muriel Patino E, Perez Perez E, Moreno Prat M
[Acute intermittent porphyria and chronic transaminase elevation].
Gastroenterol Hepatol. 2008; 31: 225-8
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Acute intermittent porphyria is an autosomal dominant inherited disorder resulting from a deficiency of porphobilinogen deaminase activity, the third enzyme in the heme biosynthesis pathway. This disease is uncommon, although the prevalence is higher in asymptomatic heterozygotic carriers; however, this prevalence is difficult to establish because of the absence of symptoms. Although acute intermittent porphyria is a multisystemic disease, its most common form of presentation is abdominal pain and neurological or mental symptoms, which can sometimes be due to precipitating factors such as reduced energy intake, smoking, alcohol, some drugs, and stress. Diagnosis can be made by testing urinary porphobilinogen levels, with subsequent measurement of enzyme activity and DNA testing. Treatment is based on prevention of porphyria attacks by avoiding precipitating factors and early administration of intravenous glucose or hemin therapy. We present the case of a patient diagnosed with acute intermittent porphyria based on study of chronic mild alanine aminotransferase (ALT) elevation.

Xu LR, Yan X, Luo M, Guan YX, Yao SJ
Preparation, characterization and refolding in vitro of a recombinant human cyclophilin A mutant: effect of a single Pro/Ser substitution on cyclophilin A structure and properties.
Biotechnol Prog. 2008; 24: 302-10
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A recombinant cyclophilin A (CypA) mutant, which carries a serine instead of proline at sequence 16, was prepared for structural and functional assessment for human CypA. Soluble expression of the recombinant CypA mutant in E. coli was obtained under 30 degrees C, 180 rpm culture condition after being induced by IPTG. Ion exchange chromatography was used to purify the CypA mutant in a single step, and a high activity recovery of target protein with a high purity was achieved. Peptide fragments produced by trypsin proteolysis were applied to MALDI-TOF-MS, and searching results from the NCBI protein databank confirmed the protein attribution as well as the mutation sequence. Peptidyl-prolyl cis-trans isomerase activity was assayed for the CypA mutant using tetrapeptide substrate Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and the calculated kcat/Km value was 1.5 x 106 M-1 s-1 at 10 degrees C, which was 10-fold lower than the previously reported constant for wild-type CypA. An Eyring plot was also carried out. Inhibition by cyclosporine A demonstrated that the IC50 value was 26.5 nM. Meanwhile the expected enhancement of intrinsic tryptophan fluorescence was quenched by the mutation. The effect of CypA mutant on accelerating protein refolding in vitro was investigated in ribonuclease A refolding process, and it was found that 10% slow phase could be catalyzed by CypA. The protein was subject to urea and GdmCl denaturation, where both activity and fluorescence served as structural probes. Activity recovery indicated this CypA mutant was extremely sensitive to GdmCl and the susceptibility to urea was increased. Low pH could also destabilize CypA. Furthermore the refolding of this CypA mutant itself was studied. Although the activity yield was nearly unchanged, the former proposed folding/assembly pathway might be altered. Fluorescence chart also demonstrated that the folding time was extended, and fast-folding and slow-folding analysis indicated the slow-folding rate constant presented a concentration dependence property denoting the autocatalysis of the foldase.

Billoo AG, Lone SW
A family with acute intermittent porphyria.
J Coll Physicians Surg Pak. 2008; 18: 316-8
Display abstract
Porphyrias are inherited defects in heme metabolism that result in excessive secretion of porphyrins and porphyrin precursors. Porphyrias can be classified into acute, (neuropsychiatric), cutaneous and mixed forms. There are seven main types of porphyrias; acute intermittent porphyria and plumboporphyria are predominantly neuropsychiatric; congenital erythropoietic porphyria, porphyria cutanea tarda and erythropoietic protoporphyria have predominantly cutaneous manifestations and hereditary coproporphyria and variegate porphyria are classified as mixed as they both have neuropsychiatric and cutaneous features. They cause life-threatening attacks of neurovisceral symptoms that mimic many other acute medical and psychiatric conditions. Lack of clinical recognition often delays effective treatment, and inappropriate diagnostic tests may lead to misdiagnosis and inappropriate treatment. Although the specific enzyme and gene defect have been identified, diagnosis and treatment of these disorders present formidable challenges because their signs and symptoms mimic other common conditions. We present a case report of a 13 years old girl who suffers from acute intermittent porphyria and the family tree showing all members who suffer from it.

Esra Onen F et al.
Design, synthesis and evaluation of potent thymidylate synthase X inhibitors.
Bioorg Med Chem Lett. 2008; 18: 3628-31
Display abstract
Three synthesized series of compounds based on a thiazolidine core allowed identification of potent inhibitors of thymidylate synthase X. The evaluation of the catalytic activity of the enzyme in the presence of these molecules revealed two distinct classes of compounds that inhibit ThyX with submicromolar concentrations, which could lead, after optimization, to effective inhibitors with potential biomedical interest.

Chen Y, Zhao JX, Liu JW, Cui J, Li L, Tian W
Lack of association of delta-aminolevulinic acid dehydratase genotype with blood lead levels in environmentally exposed children of Uygur and Han populations.
Acta Paediatr. 2008; 97: 1717-20
Display abstract
AIM: A cross-section study was conducted to explore the association between polymorphism of delta-aminolevulinic acid dehydratase (ALAD) and lead poisoning in Uygur and Han children in China. METHODS: The ALAD genotyping was determined by PCR-RFLP in 443 Uygur and 469 Han children aged 6-10 years from Urumqi in Xinjiang province. RESULTS: The blood lead levels of 912 environmentally exposed children ranged from 0.5 to 48.2 microg/dL, with a mean of 5.45 microg/dL and a standard deviation of 0.22 microg/dL, and 23. Thirty-one percent individuals were with blood lead level > or =10 microg/dL. The mean and standard deviation of blood lead levels were 5.57 +/- 0.223 microg/dL and 5.30 +/- 0.224 microg/dL in Uygur and Han children, respectively. The frequencies of the allele ALAD1 and ALAD2 in Uygur subjects were 90.52% and 9.48%, and in Han subjects were 95.73% and 4.27%, respectively (chi-square = 19.55, p < 0.05). No statistic correlation between the distribution of ALAD alleles and the blood lead level was found in both populations. CONCLUSION: A significant difference was seen in the frequency distribution of ALAD genotype between the different races. The genetic susceptibility of ALAD polymorphism to lead toxicity may exhibit in a lead dose-dependent manner.

Delev D et al.
Modelling and expression studies of two novel mutations causing factor V deficiency.
Thromb Haemost. 2008; 100: 766-72
Display abstract
Human coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations, Tyr91Asn and Asp2098Tyr, found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation. Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu(2+) binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.

Crowhurst KA, Mayo SL
NMR-detected conformational exchange observed in a computationally designed variant of protein Gbeta1.
Protein Eng Des Sel. 2008; 21: 577-87
Display abstract
Detailed biophysical characterization of computationally designed proteins has become increasingly important in order to thoroughly understand the properties of these variants compared with wild-type and to apply this knowledge to future designs. The protein dynamics and structural properties of a computationally designed variant (Delta1.5) of the beta1 domain of streptococcal protein G (Gbeta1) were measured using multinuclear NMR methods. Results from relaxation, diffusion and hydrogen exchange experiments indicate that the variant weakly self-associates at NMR concentrations, with evidence for multiple binding sites. Although comparison of fast (ps-ns) timescale motions shows only small differences in dynamics between Delta1.5 and wild-type, results from the measurement of intermediate (mus-ms) timescale motions are very different. Significant backbone conformational exchange has been observed in the variant at positions all along the sequence, whereas the wild-type Gbeta1 shows little evidence for this type of motion. This increased conformational exchange in Delta1.5 has been attributed to core overpacking resulting from the incorporation of two large hydrophobic side chains and the loss of an aromatic T-stacking interaction. These data highlight, in detail, the potential consequences of incorporating major perturbations in the core of a protein and the need to carry out more detailed analyses of the biophysical properties of designed proteins in order to better understand and predict the effects of mutations.

Golombieski RM, Graichen DA, Pivetta LA, Nogueira CW, Loreto EL, Rocha JB
Diphenyl diselenide [(PhSe)2] inhibits Drosophila melanogaster delta-aminolevulinate dehydratase (delta-ALA-D) gene transcription and enzyme activity.
Comp Biochem Physiol C Toxicol Pharmacol. 2008; 147: 198-204
Display abstract
The main objective of the present study was to compare the inhibitory effect of diphenyl diselenide (PhSe)(2) and Pb(2+) on mice and fruit fly delta-Aminolevulinate dehydratase (delta-ALA-D). Optimum pH was quite different for mice (pH 6.5) and flies (pH 8.5). At pH 8.5, the inhibitory potency of (PhSe)(2) was higher for the fruit flies (IC(50) 8.2 micromol/l) than for mice (IC(50) 19.5 micromol/l). Pb(2+) inhibited mice delta-ALA-D at pH 6.5 (IC(50) 6.2 micromol/l) and 8.5 (IC(50) 5.6 micromol/l) with higher potency than the fly enzyme (IC(50) 43.7 micromol/l). delta-ALA-D transcription was reduced by 15% in flies exposed to 0.3 mmol/kg (PhSe)(2), which is similar to the reduction observed in activity measured in the presence of dithiothreitol. The three-dimensional prediction by SWISS-PROT mouse and fly delta-ALA-D revealed differences in the number of hydrogen bonds and turns for the 2 enzymes. Sulfhydryl groups (-SH) that could be oxidized by (PhSe)(2) are conserved in the two sources of enzyme. Distinct responsiveness to pH, (PhSe)(2) and Pb(2+) of these enzymes may be related to subtle differences in tertiary or quaternary structure of mouse and fly delta-ALA-D. Furthermore, mechanism underlying enzyme inhibition after in vivo exposure seems to be different for Drosophila melanogaster and rodent enzymes.

Hsi G, Cullen LM, Macintyre G, Chen MM, Glerum DM, Cox DW
Sequence variation in the ATP-binding domain of the Wilson disease transporter, ATP7B, affects copper transport in a yeast model system.
Hum Mutat. 2008; 29: 491-501
Display abstract
ATP7B is a copper transporting P-type ATPase defective in the autosomal recessive copper storage disorder, Wilson disease (WND). Functional assessment of variants helps to distinguish normal from disease-causing variants and provides information on important amino acid residues. A total of 11 missense variants of ATP7B, originally identified in WND patients, were examined for their capacity to functionally complement a yeast mutant strain in which the yeast gene ortholog, CCC2, was disrupted. Solution structures of ATP7B domains were used to predict the effects of each variant on ATP7B structure. Three variants lie within the copper-binding domain and eight within the ATP-binding domain of ATP7B. All three ATP7B variants within the copper-binding domain and four within the ATP-binding domain showed full complementation of the yeast ccc2 phenotype. For the remaining four located in the ATP-binding domain, p.Glu1064Lys and p.Val1106Asp were unable to complement the yeast ccc2 high-affinity iron uptake deficiency phenotype, apparently due to mislocalization and/or change in conformation of the variant protein. p.Leu1083Phe exhibited a temperature-sensitive phenotype with partial complementation at 30 degrees C and a severe deficit at 37 degrees C. p.Met1169Val only partially complemented the ccc2 phenotype at 30 degrees C and 37 degrees C. Therefore, four variant positions were identified as important for copper transport and as disease-causing changes. Since the yeast assay specifically evaluates copper transport function, variants with normal transport could be defective in some other aspect of ATP7B function, particularly trafficking in mammalian cells. Functional assessment is critical for reliable use of mutation analysis as an aid to diagnosis of this clinically variable condition.

Pedersen M, Lo Leggio L, Grossmann JG, Larsen S, Hammer K
Identification of quaternary structure and functional domains of the CI repressor from bacteriophage TP901-1.
J Mol Biol. 2008; 376: 983-96
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The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein CI, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P(L) and the lysogenic promoter P(R) by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of CI without destroying the ability of CI to repress transcription from the P(L) or the bistable switch properties. We showed that a helix-turn-helix motif located in the N-terminal part of CI is involved in DNA binding by introducing specific point mutations. Purification of CI and truncated forms of CI followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of CI was required for oligomerization and that CI may exist as a hexamer in solution. Furthermore, expression and purification of the C-terminal part of CI (amino acids 92-180) showed that this part of the protein contained all the amino acids required to form an oligomer with an apparent molecular weight corresponding to a hexamer. We found that the C-terminal end of CI was required for de-repression of the P(L) following SOS induction, suggesting that the hexameric form of CI is needed for this or that this part of the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably a trimer of dimers.

Rivolta CM et al.
Two compound heterozygous mutations (c.215delA/c.2422T-->C and c.387delC/c.1159G-->A) in the thyroid peroxidase gene responsible for congenital goitre and iodide organification defect.
Clin Endocrinol (Oxf). 2007; 67: 238-46
Display abstract
BACKGROUND: Iodide organification defects are frequently but not always associated with mutations in the thyroid peroxidase (TPO) gene and characterized by a positive perchlorate discharge test. These mutations phenotypically produce a congenital goitrous hypothyroidism, with an autosomal recessive mode of inheritance. OBJECTIVES: In the present study we extended our initial molecular studies in six unrelated patients heterozygous for the TPO mutations, in order to identify the second mutation in this autosomal recessive disease. METHODS: The promoter and the complete coding regions of the human TPO and DUOXA2 genes, along with the flanking regions of each intron were analysed by direct DNA sequencing. RESULTS: Four different inactivating TPO mutations were identified in two patients: two novel mutations (c.215delA [p.Q72fsX86] and c.1159G-->A [p.G387R]) and two previously reported (c.387delC [p.N129fsX208] and c.2422T-->C [p.C808R]), confirming the inheritance of two different compound heterozygous mutations, c.215delA/c.2422T-->C and c.387delC/c.1159G-->A. The remaining four patients did not show additional inactivating mutations in the TPO gene and all had only the wild type sequencing in the DUOXA2 gene. CONCLUSIONS: We have reported two patients with iodide organification defect caused by two compound heterozygous mutations, c.215delA/c.2422T-->C [p.Q72fsX86/p.C808R] and c.387delC/c.1159G-->A [p.N129fsX208/p.G387R], in the TPO gene and four patients with monoallelic TPO defect. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of congenital hypothyroidism.

de Figueiredo RM, Coudray L, Dubois J
Synthesis and biological evaluation of potential bisubstrate inhibitors of protein farnesyltransferase. Design and synthesis of functionalized imidazoles.
Org Biomol Chem. 2007; 5: 3299-309
Display abstract
A novel series of compounds, derived from 2,5-functionalized imidazoles, have been synthesized as potential bisubstrate inhibitors of protein farnesyltransferase (FTase) using structure-based design. These compounds have a 1,4-diacid chain and a tripeptide connected by an imidazole ring. The synthetic strategy relies on the functionalization at the C-2 position of the heterocycle with the diacid side chain and peptide coupling at the C-5 position. Several new compounds were synthesized in good yields. Kinetic experiments on the most active compounds revealed different binding modes depending on the diacid chain length.

Kadam RU, Chavan A, Roy N
Pharmacophoric features of Pseudomonas aeruginosa deacetylase LpxC inhibitors: an electronic and structural analysis.
Bioorg Med Chem Lett. 2007; 17: 861-8
Display abstract
Various electronic properties of structurally diverse synthetic LpxC inhibitors containing oxazoline, aroylserine and thiazoline rings were calculated and correlated with biological activity. These electronic features include the magnitude and locations of 3-dimensional molecular electrostatic potentials, hydrogen bond acceptor/donor density, lowest unoccupied molecular orbital, and highest occupied molecular orbital. Strong correlation of these stereo-electronic properties with LpxC inhibitory potency reveals the potential pharmacophoric features of specific LpxC inhibitors. Thus, these pharmacophoric features of LpxC inhibitors based on electronic and surface analysis could be successfully exploited for designing more potent LpxC inhibitors.

Zhang Y, Jin G, Illarionov B, Bacher A, Fischer M, Cushman M
A new series of 3-alkyl phosphate derivatives of 4,5,6,7-tetrahydro-1-D-ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as inhibitors of lumazine synthase: design, synthesis, and evaluation.
J Org Chem. 2007; 72: 7176-84
Display abstract
Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products. All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase, with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues bind similarly.

Wen X et al.
Pentacyclic triterpenes. Part 5: synthesis and SAR study of corosolic acid derivatives as inhibitors of glycogen phosphorylases.
Bioorg Med Chem Lett. 2007; 17: 5777-82
Display abstract
The synthesis and biological evaluation of corosolic acid derivatives and related compounds as inhibitors of rabbit muscle glycogen phosphorylase a is described. Within this series of compounds, 8 (IC(50)=7.31 microM), 12d (IC(50)=3.26 microM), and 12e (IC(50)=5.1 microM) exhibited more potent activities than the parent compound 1 (IC(50)=20 microM). SAR of these compounds is also discussed.

Teruel Sanchez-Vegazo C, Maillo Fernandez L, Martinez-Fidalgo Vazquez C, Boixeda de Miguel D
[Doss porphyria without demonstrated enzyme deficit: a secondary porphyrinuria?].
Rev Clin Esp. 2006; 206: 469-70

Sassa S
Modern diagnosis and management of the porphyrias.
Br J Haematol. 2006; 135: 281-92
Display abstract
Recent advances in the molecular understanding of the porphyrias now offer specific diagnosis and precise definition of the types of genetic mutations involved in the disease. Molecular diagnostic testing is powerful and very useful in kindred evaluation and genetic counselling when a disease-responsible mutation has been identified in the family. It is also the only way to properly screen asymptomatic gene carriers, facilitating correct treatment and appropriate genetic counselling of family members at risk. However, it should be noted that DNA-based testing is for the diagnosis of the gene carrier status, but not for the diagnosis of clinical syndrome or severity of the disease, e.g. an acute attack. For the diagnosis of clinically expressed porphyrias, a logical stepwise approach including the analysis of porphyrins and their precursors should not be underestimated, as it is still very useful, and is often the best from the cost-effective point of view.

Akagi R, Kato N, Inoue R, Anderson KE, Jaffe EK, Sassa S
delta-Aminolevulinate dehydratase (ALAD) porphyria: the first case in North America with two novel ALAD mutations.
Mol Genet Metab. 2006; 87: 329-36
Display abstract
The molecular basis of the enzymatic defect responsible for delta-aminolevulinate dehydratase (ALAD) porphyria (ADP) was investigated in a 14-year-old male who presented clinical and laboratory findings typical of ADP. Nucleotide sequence analysis of ALAD cDNAs from the proband revealed two novel mutations, a 265G to A base transition (C1) and a 394C to T base transition (C2), resulting in amino acid substitutions, Glu89Lys and Cys132Arg, respectively. Both mutations were present within exon 5 of the ALAD gene, and appeared to influence the binding of zinc to the enzyme which is essential for enzyme activity. It was found that the C1 mutation was inherited from his father, while the C2 mutation was from his mother. Expression of these mutant ALAD cDNAs in Chinese hamster ovary cells produced normal ALAD mRNA levels, but markedly decreased ALAD protein and enzyme activity. These results suggest that the combination of the two aberrant ALADs with little enzyme activity accounts for the markedly decreased ALAD activity observed in the proband. This case represents the molecular analysis of the ALAD gene defects in the first case of ADP identified in North America, who is a compound heterozygote for two novel ALAD gene defects.

Bollinger JA, Brown DE, Dooley DM
The Formation of lysine tyrosylquinone (LTQ) is a self-processing reaction. Expression and characterization of a Drosophila lysyl oxidase.
Biochemistry. 2005; 44: 11708-14
Display abstract
Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.

Kooijman MM, Brand HS
Oral aspects of porphyria.
Int Dent J. 2005; 55: 61-6
Display abstract
Porphyria is a diverse group of diseases in which the biosynthesis of heme is disrupted by either genetic defects or environmental factors. This review gives an overview of the different types of porphyria and describes possible causes, clinical signs, diagnosis and therapy. In addition, the oral manifestations of porphyria and the potential implications of the disease for dental management are discussed.

Pauza NL, Cotti MJ, Godar L, Ferramola de Sancovich AM, Sancovich HA
Disturbances on delta aminolevulinate dehydratase (ALA-D) enzyme activity by Pb2+, Cd2+, Cu2+, Mg2+, Zn2+, Na+, K+ and Li+: analysis based on coordination geometry and acid-base Lewis capacity.
J Inorg Biochem. 2005; 99: 409-14
Display abstract
ALA-D (EC 4.2.1.24) is the first cytosolic enzyme in the haem metabolic pathway. Some metals compete with its major cofactor Zn(2+), modifying both enzyme structure and function. Our purpose was to contribute to the understanding of the biochemical role of metals such as Pb(2+), Cd(2+), Cu(2+), Mg(2+), Zn(2+), Na(+), K(+) and Li(+) on ALA-D, using chicken embryos as experimental model. Mg(2+) and Zn(2+) showed enzyme activation in yolk sac membrane (YSM) (113% at 10(-5) M Mg(2+) and from 10(-4) M Zn(2+)), and slight inactivation in liver. Cd(2+) and Cu(2+) caused a non allosteric inhibition in both tissues (100% from 10(-4) M). Surprisingly Pb(2+) was not such a strong inhibitor. Interference of cations during the Schiff base formation in enzymatic catalysis process is explained considering their Lewis acid-base capacity, coordination geometry and electron configuration of valence. Interactions among monovalent cations and biochemical substances are governed chiefly by its electrostatic potential. 0.1 M K(+) and 0.4 M Na(+) produced 30% of enzymatic inhibition by the interference on interactions among the functional subunits. Li(+) activated the YSM enzyme (130% at 10(-5) M) due to a more specific interaction. This study may contribute to elucidate for the first time the possible structural differences between the YSM and liver enzymes from chicken embryo.

Erskine PT et al.
Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid.
Acta Crystallogr D Biol Crystallogr. 2005; 61: 1222-6
Display abstract
The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 A. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA).

Coates L et al.
The X-ray structure of the plant like 5-aminolaevulinic acid dehydratase from Chlorobium vibrioforme complexed with the inhibitor laevulinic acid at 2.6 A resolution.
J Mol Biol. 2004; 342: 563-70
Display abstract
5-Aminolaevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyses the dimerisation of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen. ALAD from Chlorobium vibrioforme is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts a TIM-barrel fold with a 30 residue N-terminal arm extension. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact principally via their arm regions to form octamers in which each active site is located on the surface. The active site contains two invariant lysine residues (200 and 253), one of which (Lys253) forms a Schiff base link with the bound substrate analogue, laevulinic acid. The carboxyl group of the laevulinic acid forms hydrogen bonds with the side-chains of Ser279 and Tyr318. The structure was examined to determine the location of the putative active-site magnesium ion, however, no evidence for the metal ion was found in the electron density map. This is in agreement with previous kinetic studies that have shown that magnesium stimulates but is not required for activity. A different site close to the active site flap, in which a putative magnesium ion is coordinated by a glutamate carboxyl and five solvent molecules may account for the stimulatory properties of magnesium ions on the enzyme.

Sakamoto D et al.
A mutation in the gene for delta-aminolevulinic acid dehydratase (ALAD) causes hypochromic anemia in the medaka, Oryzias latipes.
Mech Dev. 2004; 121: 747-52
Display abstract
A genetic screen for mutations affecting embryogenesis in the medaka, Oryzias latipes, identified a mutant, whiteout (who), that exhibited hypochromic anemia. The who mutant initially had the normal number of blood cells, but it then gradually decreased during the embryonic and larval stages. The blood cells in the who mutants show an elongated morphology and little hemoglobin activity. Genetic mapping localized who to the vicinity of a LG12 marker, olgc1. By utilizing the highly conserved synteny between medaka and pufferfish, we identified a gene for delta-aminolevulinic acid dehydratase (ALAD), which is the second enzyme in the heme synthetic pathway, as a candidate for who. We found a missense mutation in the alad gene that was tightly linked to the who phenotype, strongly suggesting that the hypochromic anemia phenotype in the who mutant is caused by a loss of the alad function. Thus, who mutants represent a model for the human disease ALAD-deficiency porphyria.

Jaffe EK
Investigations on the metal switch region of human porphobilinogen synthase.
J Biol Inorg Chem. 2003; 8: 176-84
Display abstract
Porphobilinogen synthase (PBGS) is an ancient and highly conserved protein that functions in the first common step in tetrapyrrole biosynthesis. The PBGS protein sequence contains a unique metal switch region that has been postulated to dictate an exclusive catalytic use of either zinc or magnesium, and perhaps also potassium. In some PBGS, the cysteines of the metal switch sequence DXCXCX(Y/F)X(3)G(H/Q)CG have been demonstrated to bind a catalytic zinc, and in other PBGS, the aspartic acid residues of the metal switch sequence DXALDX(Y/F)X(3)G(H/Q)DG have been postulated to bind a catalytically essential magnesium and/or potassium. The current work describes chimeric proteins that contain the aspartate-rich sequences of pea PBGS and Pseudomonas aeruginosa PBGS in place of the naturally occurring cysteine-rich sequence of human PBGS. The resultant chimeric PBGS proteins, peainhuman PBGS and psuinhuman PBGS, are substantially activated by both magnesium and potassium, but not by zinc. The specific activities of the chimeras are significantly lower than human PBGS. Detailed kinetic and inhibition data are presented for both chimeric proteins and are discussed in terms of this unique phylogenetic variation in metal ion usage. The identity of a basic residue, which is Arg221 in human PBGS, strictly correlates with the presence or absence of the cysteine-rich sequence. Those PBGS with the aspartate-rich metal switch sequence contain Lys in the analogous position. The R221K mutation was inserted into wild type and chimeric human PBGS and found to further reduce the activity of both, illustrating the subtle nature of the role of this residue.

Tasnadi G, Bor M, Pusztai A
[Treatment of acute porphyrias. The importance of follow-up of patients and carriers].
Orv Hetil. 2003; 144: 933-8
Display abstract
Acute porphyrias are caused by the inherited decreased activity of the enzymes of the heme biosynthesis pathway. Depending on the affected enzyme there are 4 types of them: acute intermittent porphyria, porphyria variegata, coproporphyria and delta-aminolevulinic acid dehydratase deficient porphyria, listed in order of their frequency. Basically the clinical picture is the same in the four types of acute porphyria. The most frequent complaints and symptoms are: cramping abdominal pain, nausea, vomiting, muscle weakness of the limbs then, in the advanced phase, there is a red-colored urine, hyponatremia, subileus, acute psychosis and Landry-type paralysis. Without proper treatment death is caused by respiratory paralysis or serious arrhythmia. In case of suspicion of acute porphyria it is mandatory to identify the type of the acute porphyria and the actual status of the patient. The later indicates what kind of treatment should be used. In the acute phase the early therapy with heme arginate is the treatment of choice. Since the clinical symptoms are precipitated by endogenous or exogenous inducing factors--most often by drugs-, the drugs negatively affecting the heme biosynthesis should be omitted at once even in the suspicion of acute porphyria. The role of the inducing factors in the manifestation of the clinical symptoms makes possible the prevention. It is possible to avoid the inducing factors and this way to prevent the acute attack if the acute porphyrias are recognized in time and the patients and the carriers are under regular control. The patients receive special identification card and the up-to-date list of safe drugs. They can use only these drugs in any kind of illness. Other drugs should be considered as porphyrinogenic since it is impossible to predict based on their chemical structure if they negatively affect the heme biosynthesis.

Frere F et al.
Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism.
J Mol Biol. 2002; 320: 237-47
Display abstract
All natural tetrapyrroles, including hemes, chlorophylls and vitamin B12, share porphobilinogen (PBG) as a common precursor. Porphobilinogen synthase (PBGS) synthesizes PBG through the asymmetric condensation of two molecules of aminolevulinic acid (ALA). Crystal structures of PBGS from various sources confirm the presence of two distinct binding sites for each ALA molecule, termed A and P. We have solved the structure of the active-site variant D139N of the Mg2+-dependent PBGS from Pseudomonas aeruginosa in complex with the inhibitor 5-fluorolevulinic acid at high resolution. Uniquely, full occupancy of both substrate binding sites each by a single substrate-like molecule was observed. Both inhibitor molecules are covalently bound to two conserved, active-site lysine residues, Lys205 and Lys260, through Schiff bases. The active site now also contains a monovalent cation that may critically enhance enzymatic activity. Based on these structural data, we postulate a catalytic mechanism for P. aeruginosa PBGS initiated by a C-C bond formation between A and P-side ALA, followed by the formation of the intersubstrate Schiff base yielding the product PBG.

Jaffe EK et al.
Species-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid.
J Biol Chem. 2002; 277: 19792-9
Display abstract
Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGS-catalyzed reaction.

Sassa S
The porphyrias: genetic and acquired aspects.
Rinsho Ketsueki. 2001; 42: 237-41

Downey D
Porphyria: the road not traveled.
Med Hypotheses. 2001; 56: 73-6
Display abstract
As our ability to evaluate patients with porphyria expands there have been some inconsistencies that are not easily explained. These inconsistencies are biochemical, enzymatic and genetic in nature. Recent work by others has led me to believe there is another way to look at the problem. I will propose how an evolutionary approach could reconcile these problems. In doing so it will imply that porphyria could potentially affect many more people than currently thought.

Kelada SN, Shelton E, Kaufmann RB, Khoury MJ
Delta-aminolevulinic acid dehydratase genotype and lead toxicity: a HuGE review.
Am J Epidemiol. 2001; 154: 1-13
Display abstract
The ALAD gene (chromosome 9q34) codes for delta-aminolevulinic acid dehydratase (ALAD) (E.C. 4.2.1.24). ALAD catalyzes the second step of heme synthesis and is polymorphic. The ALAD G177C polymorphism yields two codominant alleles, ALAD-1 and ALAD-2, and it has been implicated in susceptibility to lead toxicity. Genotype frequencies vary by geography and race. The rarer ALAD-2 allele has been associated with high blood lead levels and has been thought to increase the risk of lead toxicity by generating a protein that binds lead more tightly than the ALAD-1 protein. Other evidence suggests that ALAD-2 may confer resistance to the harmful effects of lead by sequestering lead, making it unavailable for pathophysiologic participation. Recent studies have shown that individuals who are homozygous for the ALAD-1 allele have higher cortical bone lead levels; this implies that they may have a greater body lead burden and may be at higher risk of the long-term effects of lead. Individuals exposed to lead in occupational settings have been the most frequent subjects of study. Genotype selection bias may limit inferences from these studies. No firm evidence exists for an association between ALAD genotype and susceptibility to lead toxicity at background exposure levels; therefore, population testing for the ALAD polymorphism is not justified.

Lim HW
PORPHYRIA UPDATE.
Pediatr Dermatol. 2000; 17: 75-83

Jarret C et al.
Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.
Chem Biol. 2000; 7: 185-96
Display abstract
BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Zheng R, Blanchard JS
Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue.
Biochemistry. 2000; 39: 16244-51
Display abstract
Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.

Akagi R, Shimizu R, Furuyama K, Doss MO, Sassa S
Novel molecular defects of the delta-aminolevulinate dehydratase gene in a patient with inherited acute hepatic porphyria.
Hepatology. 2000; 31: 704-8
Display abstract
Cloning and expression of the defective gene for delta-aminolevulinate dehydratase (ALAD) from the second of 2 German patients with ALAD deficiency porphyria (ADP), who had been originally reported by Doss et al. in 1979, were performed. Cloning of cDNAs for the defective ALAD were performed using Epstein-Barr virus (EBV)-transformed lymphoblastoid cells of the proband, and nucleotide sequences of cloned cDNA were determined. Two separate mutations of ALAD cDNA were identified in each ALAD allele. One was G457A, termed "H1," resulting in V153M substitution, while the other was a deletion of 2 sequential bases at T(818) and C(819), termed "H2," resulting in a frame shift with a premature stop codon at the amino acid position of 294. Using allele-specific oligonucleotide hybridization, the mother of the proband was shown to have an H1 defect, while using genomic DNA analysis, the father was shown to have an H2 defect. Expression of H1 cDNA in Chinese hamster ovary cells produced an ALAD protein with only a partial activity (10.65% +/- 1.80% of the normal), while H2 cDNA encoded no significant protein. These data thus demonstrate that the proband was associated with 2 novel molecular defects of the ALAD gene, 1 in each allele, and account for the extremely low ALAD activity in his erythrocytes ( approximately 1% of normal).

Gross M, Hessefort S, Olin A
Purification of a 38-kDa protein from rabbit reticulocyte lysate which promotes protein renaturation by heat shock protein 70 and its identification as delta-aminolevulinic acid dehydratase and as a putative DnaJ protein.
J Biol Chem. 1999; 274: 3125-34
Display abstract
We reported recently that a rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory and p60 and hop by others) functions as a hsp 70 recycling protein and markedly enhances the renaturation of luciferase by hsp 70 (Gross, M., and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70. ADP to hsp 70.ATP, thus enhancing the protein folding activity of hsp 70, is caused by the purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein contaminant. To determine the relationship between RF-hsp 70 and the DnaJ/hsp 40 heat shock protein family, which also enhances protein renaturation by hsp 70, we purified a 38-kDa protein from rabbit reticulocyte lysate based upon its ability to stimulate renaturation of luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa protein has indicated, unexpectedly, that it is the enzyme delta-aminolevulinic acid dehydratase (ALA-D) and that it does not contain detectable sequences corresponding to the DnaJ/hsp 40 protein family. In addition, immunoblot analysis with a polyclonal antibody made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D is about as active in converting delta-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D purified from other sources. Rabbit reticulocyte ALA-D stimulates the renaturation of luciferase by hsp 70 up to 10-fold at concentrations that are the same as or less than that of hsp 70, and it has no renaturation activity in the absence of hsp 70. The renaturation effect of ALA-D is additive with that of RF-hsp 70 at limiting or saturating concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote the dissociation of hsp 70.ADP in the presence of ATP. The renaturation-enhancing effect of ALA-D may be caused by a region near its carboxyl terminus which has sequence homology to the highly conserved domain of the DnaJ protein family, which is similar to the sequence homology between this domain and a carboxyl-terminal region in auxilin, a DnaJ-like protein that requires this region for its hsp 70-dependent function (Ungewickell, E., Ungewickell, H., Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).

Frankenberg N, Jahn D, Jaffe EK
Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.
Biochemistry. 1999; 38: 13976-82
Display abstract
Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.

Erskine PT et al.
X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution.
Biochemistry. 1999; 38: 4266-76
Display abstract
5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic acid to form the pyrrole, porphobilinogen. ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact, principally via their arm regions, to form octamers in which each active site is located on the surface. The active site contains two lysine residues (195 and 247), one of which (Lys 247) forms a Schiff base link with the bound substrate analogue, levulinic acid. Of the two substrate binding sites (referred to as A and P), our analysis defines the residues forming the P-site, which is where the first ALA molecule to associate with the enzyme binds. The carboxyl group of the levulinic acid moiety forms hydrogen bonds with the side chains of Ser 273 and Tyr 312. In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule. We infer that the second substrate binding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety. Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate. Another metal binding site, close to the active site flap, in which a putative zinc ion is coordinated by a carboxyl and five solvent molecules may account for the activating properties of magnesium ions.

Shimoni-Livny L et al.
Crystallization and preliminary X-ray diffraction studies of E. coli porphobilinogen synthase and its heavy-atom derivatives.
Acta Crystallogr D Biol Crystallogr. 1998; 54: 438-40
Display abstract
Porphobilinogen synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 A) are reported. These crystals diffract to about 3.0 A resolution.

Norton E, Sarwar M, Shoolingin-Jordan P
Mechanistic studies on E.coli 5-aminolaevulinic acid dehydratase.
Biochem Soc Trans. 1998; 26: 285-285

Carrell RW, Lomas DA
Conformational disease.
Lancet. 1997; 350: 134-8
Display abstract
Several diverse disorders, including the prevalent dementias and encephalopathies, are now believed to arise from the same general disease mechanism. In each, there is abnormal unfolding and then aggregation of an underlying protein. The gradual accumulation of these aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the clinical disease. The understanding of these processes at the molecular level is opening prospects of more rational approaches to investigation and therapy.

Schreiber WE
A molecular view of the neurologic porphyrias.
Clin Lab Med. 1997; 17: 73-83
Display abstract
At the present time, the use of molecular techniques for diagnosing the neurologic porphyrias is limited mainly to those laboratories doing research in the area. Because any of four genes may be involved, it is important to confirm the diagnosis in the index case before undertaking molecular investigation. With advances in methodology, it may eventually become possible to test for the many mutations that cause these disorders in a simple, cost-effective manner.

Valentini G et al.
An essential lysine in the substrate-binding site of ornithine carbamoyltransferase.
Eur J Biochem. 1996; 239: 397-402
Display abstract
Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

Guolo M, Stella AM, Melito V, Parera V, Del C Batlle AM
Altered 5-aminolevulinic acid metabolism leading to pseudoporphyria in hemodialysed patients.
Int J Biochem Cell Biol. 1996; 28: 311-7
Display abstract
Hemodialysed patients with no history of porphyria may present neurological symptoms similar to those seen in acute porphyrias. Porphyria has been associated with an increase in plasma levels of 5-aminolevulinic acid and porphobilinogen. Our aim was to evaluate these parameters and the activities of the enzymes involved in the first steps of heme metabolism in non-porphyric hemodialysed patients. The activities of 5-aminolevulinate dehydratase and deaminase were determined in red blood cells (RBC) from 78 hemodialysed patients, before and after dialysis. Plasma levels of 5-aminolevulinic acid, porphobilinogen and zinc were also measured. These parameters were also measured in 40 volunteers to obtain controls levels. The levels of 5-aminolevulinic acid (0.98 +/- 0.09 microgram/ml) and porphobilinogen (1.32 +/- 0.13 micrograms/ml) were raised in non-porphyric patients prior to hemodialysis (P < 0.001) compared with controls (5-aminolevulinic acid 0.13 +/- 0.02 microgram/ml; porphobilinogen 0.90 +/- 0.09 microgram/ml). After dialysis there was a decrease in both 5-aminolevulinic acid (to 0.61 +/- 0.05 microgram/ml) and porphobilinogen (to 1.10 +/- 0.16 micrograms/ml) although both parameters remained higher than controls (P < 0.001). The activities of both 5-aminolevulinate dehydratase (0.550 +/- 0.095 U/ml RBC), and deaminase (54.13 +/- 9.13 U/ml RBC) were diminished in blood samples of patients before dialysis (P < 0.001) compared to controls (dehydratase 0.975 +/- 0.115 U/ml RBC; deaminase 77.32 +/- 10.00 U/ml RBC). After dialysis 5-aminolevulinate dehydratase activity was partially recovered (to 0.666 +/- 0.100 U/ml RBC) while deaminase returned to normal values (73.45 +/- 9.46 U/ml RBC). The plasma zinc concentration in hemodialysed patients (44 +/- 12 micrograms/100 ml) was significantly lower than controls (105 +/- 30 micrograms/100 ml, P < 0.001). Addition of 22.5 mM zinc to the dehydratase reaction mixture raised the activity of 5-aminolevulinate dehydratase in blood samples of hemodialysed patients taken before and after dialysis. The study reports a partial loss of activity of 5-aminolevulinate dehydratase and deaminase activities in red blood cells from non-porphyric patients undergoing hemodialysis. Since plasma zinc levels were below normal in hemodialysed patients, and the activity of 5-aminolevulinate dehydratase could be restored by the addition of zinc, it is suggested that these abnormalities in heme metabolism may be explained by altered zinc and associated antioxidant status following dialysis.

Muraoka A, Suehiro I, Fujii M, Murakami K
delta-Aminolevulinic acid dehydratase deficiency porphyria (ADP) with syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in a 69-year-old woman.
Kobe J Med Sci. 1995; 41: 23-31
Display abstract
delta-Aminolevulinic acid dehydratase deficiency porphyria (ALAD porphyria, ADP) with syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in a 69-year-old woman is reported. The patient was admitted to our hospital complaining of slight cough with low-grade fever, and treated with piperacillin sodium, resulting in complete resolution of the symptoms, following a diagnosis of bronchopneumonia. Thereafter, however, she began to complain of vomiting, abdominal pain, facial numbness and paresis of the extremities with gait disturbance, and became comatose with hyponatremia (serum Na concentration 119 mEq/L) in a few days. Laboratory tests revealed an antidiuretic hormone (ADH) level of 13.5 pg/mL, plasma osmolality 218 mOsm/KgH2O, urinary osmolality 429 mOsm/KgH20, urinary Na concentration > 20 mEq/L, and no abnormalities of thyroid, adrenal or renal function. Neither edema nor dehydration was evident. These data indicated the presence of SIADH. No abnormalities suggestive of malignant or infectious diseases such as lung cancer, pneumonia and Guillain-Barre syndrome were evident from laboratory and roentgenographic findings. As the cause of SIADH, therefore, porphyria was suspected. Metabolites and activities of enzymes in the heme biosynthetic pathway were examined, and very low activity of delta-aminolevulinic acid dehydratase (ALA-D) (0.14 mumol PBG/mL RBC/h) was found. The patient was neither an alcoholic nor a heavy smoker, and she had no past history of heavy metal intoxication, photosensitivity or tyrosinemia. On the basis of these data and clinical features, she was diagnosed as having ADP. We consider this to be the first case of ADP reported in Japan.

Fujita H, Ishida N, Akagi R
[delta-Aminolevulinate dehydratase deficiency].
Nihon Rinsho. 1995; 53: 1408-17
Display abstract
delta-Aminolevulinate dehydratase (ALAD: E. C. 4.2.1.24), the second enzyme in the heme biosynthetic pathway, condenses two moles of delta-aminolevulinic acid to form porphobilinogen. ALAD deficiency is well known to develop signs and symptoms of typical hepatic porphyria, and classified into three categories as follows: (i) ALAD porphyria, a genetic defect of the enzyme, (ii) tyrosinemia type I, a genetic defect of fumarylacetoacetase in the tyrosine catabolic pathway, producing succinylacetone (a potent inhibitor of ALAD), and (iii) ALAD inhibition by environmental hazards, such as lead, trichloroethylene, and styrene. In the present article, we will describe molecular and biochemical mechanisms to cause the enzyme defect to discuss the significance of ALAD defect on human health.

Markham GD, Myers CB, Harris KA Jr, Volin M, Jaffe EK
Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.
Protein Sci. 1993; 2: 71-9
Display abstract
The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L. J., Grunwald, J., Hankovszky, H. O., & Hideg, K., 1982, Anal. Biochem. 119, 450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (delta m = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions show that at least two of the unpaired electrons are within at most 7.6 A of each other. SL-MMTS-modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added.(ABSTRACT TRUNCATED AT 250 WORDS)

Hassoun A, Verstraeten L, Mercelis R, Martin JJ
Biochemical diagnosis of an hereditary aminolaevulinate dehydratase deficiency in a 63-year-old man.
J Clin Chem Clin Biochem. 1989; 27: 781-6
Display abstract
Porphyrin metabolism was investigated in a 63-year-old male patient who developed a subacute onset polyneuropathy with predominance of motor signs in the upper limb. The screening for lead, cadmium, mercury, aluminum and thallium was negative. The study of porphyrin metabolism showed remarkable abnormalities, particularly a very high level of plasmatic 5-aminolaevulinic acid contrasting with a normal level of porphobilinogen and a nearly complete loss of activity of aminolaevulinic acid dehydratase with no regenerative response to dithiothreitol or zinc ions. The other causes of aminolaevulinic acid dehydratase deficiency (tyrosinaemia, alcoholism, smoking, cirrhosis, renal insufficiency, diabetes mellitus) were ruled out. The diagnosis of primary aminolaevulinic acid dehydratase deficiency was proposed and confirmed by the familial study, which revealed the existence of several heterozygous members in this family.

Zapata GA et al.
Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment.
J Biol Chem. 1988; 263: 8150-6
Display abstract
Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.

Gitlin G, Bayer EA, Wilchek M
Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.
Biochem J. 1987; 242: 923-6
Display abstract
Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

Labbe RF, Bird TJ
More on identifying inherited deficiency of porphobilinogen synthase.
Clin Chem. 1985; 31: 162-162

Herndon CS, Hartman FC
2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide.
J Biol Chem. 1984; 259: 3102-10
Display abstract
A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus of the enzyme. The lack of reactivity with the spinach enzyme is explained by the deletion of the histidyl residue and the replacement of cysteine by tryptophan in the eukaryotic species. Although the nonconservation of the modified residues argues against a functional role other than maintenance of structural integrity, the extensive homology in this region among seven different species of carboxylase is compatible with the region comprising a portion of the active site.

Tschudy DP, Hess RA, Frykholm BC
Inhibition of delta-aminolevulinic acid dehydrase by 4,6-dioxoheptanoic acid.
J Biol Chem. 1981; 256: 9915-23

Harrison RA, Thomas ML, Tack BF
Sequence determination of the thiolester site of the fourth component of human complement.
Proc Natl Acad Sci U S A. 1981; 78: 7388-92
Display abstract
The fourth component of complement (C4) is inactivated by treatment with methylamine. This property is shared wit the third component (C3) and with alpha 2-macroglobulin. In each instance, the reaction with methylamine is stoichiometric, covalent, and accompanied by the appearance of a thiol group. These data are consistent with the presence of an internal thiolester bond. Incubation of C4 with [14C] methylamine in the presence of activated thiol-Sepharose resulted in immobilization of the protein via its active-site thiol. Analysis of bound C4 indicated incorporation of 1.12 mol of [14C]methylamine per mol of protein. Digestion of the immobilized protein with porcine elastase resulted in the release of C4 beta- and gamma-chains and lower molecular weight fragments. The 14C label, however, was retained on the Sepharose beads. Subsequent release of bound material with L-cysteine indicated that the radiolabel was associated with two polypeptides of Mr 25,000 [C4d(ela25)]. The released material was dialyzed and the active-site thiol was radioalkylated with iodo[2-3H]acetic acid. C4d(ela25) was further purified by chromatography on Sephadex G-100 and, after reduction and alkylation, on Sepharose CL-6B in 0.2% NaDodSO4. The C4d(ela25) pool, containing 0.83 mol of [14C]methylamine per mol of iodo[2-3H]acetic acid, was subjected to automated sequence analysis. S-carboxy-[3H]methylcysteine was released at step 21 and gamma-glutamyl-[14C]methylamide was released at step 24. The recovery of radiolabel at positions 21 and 24 confirmed the originally calculated 14C/3H incorporation ratio and further indicated that the radiolabels were present at single sites in the C4 molecule. Comparison of the derived primary structure for the thiolester site in C4 with those for the corresponding regions in C3 and alpha 2-macroglobulin has shown sequence identity. Further comparisons among these three proteins have indicated additional homologies on both the NH2- and COOH-terminal sides of the thiolester site.

Battistuzzi G, Petrucci R, Silvagni L, Urbani FR, Caiola S
delta-Aminolevulinate dehydrase: a new genetic polymorphism in man.
Ann Hum Genet. 1981; 45: 223-9
Display abstract
A method has been developed for the electrophoretic and quantitative analyses of human red cell delta-aminolevulinate dehydrase (ALADH). The enzyme is under the control of an autosomal gene, with two common codominant alleles. ALADH1 and ALADH2, with frequencies of 0.89 and 0.11, respectively, in the Italian population. Mean phenotypic enzyme activities are nearly identical: 52,. 49 and 55 mIU/g Hb for ALADH 1, 2-1 and 2 phenotypes respectively.

Hochschwender SM, Laursen RA
The lysine binding sites of human plasminogen. Evidence for a critical tryptophan in the binding site of kringle 4.
J Biol Chem. 1981; 256: 11172-6
Display abstract
Chemical modification of human degraded form of plasminogen with NH2-terminal lysine (Lys-plasminogen) and the elastase fragments kringle 1 + 2 + 3 and kringle 4 with the tryptophan reagent [14C]dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide results in the incorporation of label and the parallel loss of lysine binding ability. In the case of kringle 4, only one-half of the lysine binding sites could be inactivated, but the modified and unmodified forms could be separated by affinity chromatography. The modified form contained 1 mol of 2-hydroxy-5-nitrobenzyl groups/mol of kringle 4 and did not bind to lysine-Sepharose. Lysine analogs such as 6-aminohexanoic acid protected kringle 4 against modification. Peptide-mapping studies on this form showed that essentially all of the label was in two chymotryptic peptides containing a tryptophan corresponding to Trp426 in the plasminogen sequence. Competition experiments with anti-kringle 4 antibodies having an affinity for the lysine binding site showed that the binding of 2-hydroxy-5-nitrobenzyl-kringle 4 to antibodies was about 10 times weaker than for unmodified kringle 4. These results indicate that the integrity of specific tryptophan residue is critical to the binding of lysine and related amino acids to kringle 4of human plasminogen.

Seehra JS, Jordan PM
5-Aminolevulinic acid dehydratase: alkylation of an essential thiol in the bovine-liver enzyme by active-site-directed reagents.
Eur J Biochem. 1981; 113: 435-46
Display abstract
1. 5-Aminolevulinic acid dehydratase from bovine liver has been shown to be inactivated by 5-halolevulinic acids and 3-halolevulinic acids. 2. The substrate, 5-aminolevulinic acid, protects the enzyme from modification by 5-halolevulinic acids. 3. Using tritiated chlorolevulinic acids, it was shown that four of the subunits in the octameric enzyme are preferentially modified. 4. The susceptible enzyme group modified is an --SH group of a reactive cysteine at or near the active site. 5. Oxidized enzyme is not affected by either 5-chlorolevulinic acid or 3-chlorolevulinic acid. 6. Evidence is presented which suggests that 5-chlorolevulinic acid is acting as an active-site-directed reagent.

Liedgens W, Grutzmann R, Schneider HA
Highly efficient purificaton of the labile plant enzyme 5-aminolevulinate dehydratase (EC 4.2.1.24) by means of monoclonal antibodies.
Z Naturforsch C. 1980; 35: 958-62
Display abstract
5-Aminolevulinate dehydratase (ALAD) from spinach (Spinatia oleracea) was isolated by affinity purification on an immunoabsorbens with a yield of 70 to 80% of the activity in the crude enzyme preparation. The enzyme eluted from the immunoabsorbens was pure as judged by polyacrylamide gel electrophoresis and is a hexamer with a subunit molecular weight of about 50 000. Enzyme bound to the immunoabsorbens was able to synthesize porphobilinogen in a continuous manner. Owing to the lability of the enzyme and its low abundance in plant tissue, we have been unable to obtain similar yields of purified enzyme using classical purification procedures. This highly efficient purification was made possible by using monoclonal antibodies as described by Kohler and Milstein (Nature 256, 495 (1975)). The availability of monoclonal antibodies meant that it was not necessary to purify the enzyme to homogeneity by classical means in order to raise an antiserum specific for ALAD. Sixteen clones of cells producing antibodies against ALAD were selected. They all expressed a chi light chain but differed in the heavy chain class which was eigher gamma 1 or gamma 2a. The availability of pure ALAD enzyme and of highly specific antibodies against the enzyme now enables us to answer questions concerning properties, localization, intercellular transport and evolution of ALAD. It is clear that the technique used and the questions asked are not restricted to ALAD.

Liubchenko PN, Gladyshev BN, Ostrum IuZ, Avramenko MM
[Features of porphobilinogen synthesis from delta-aminolevulinic acid in the tissues of the internal organs of rats].
Biull Eksp Biol Med. 1980; 90: 730-2
Display abstract
The biosynthesis of porphobilinogen by homogenates of the lungs, heart, liver, kidneys, spleen, pancreas and small intestinal tissues was studied in 77 intact and in 68 rats with marked lead poisoning. The activity of ALA dehydratase in the lungs was decreased by 40.3%, in the kidneys by 40%, in the heart by 38.2% and was normal in homogenates of the liver, pancreas and small intestine. In spleen homogenates, the activity was increased by 26.3%. Potential mechanisms of different ALA dehydratase reactions in organs tissues of rats with lead poisoning are discussed.

Jacyszyn K
[Ecotoxins and enzymes].
Folia Med Cracov. 1980; 22: 305-12

Travis J, Johnson D
The carboxy terminal sequence of human alpha-1-proteinase inhibitor.
Biochem Biophys Res Commun. 1978; 84: 219-24

Meloche HP, Monti CT, Hogue-Angeletti RA
Identification of the bromopyruvate-sensitive glutamate within the active site of 2-keto-3-deoxygluconate-6-P aldolase.
Biochem Biophys Res Commun. 1978; 84: 589-94

Dworschack R, Tarr G, Plapp BV
Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.
Biochemistry. 1975; 14: 200-3
Display abstract
A single amino group in horse liver alcohol dehydrogenase was modified with methyl(14C)acetimidate by a differential labeling procedure. Lysine residues outside the active site were modified with ethyl acetimidate while a lysine residue in the active site was protected by the formation of an enzyme-NAD+-pyrazole complex. After the protecting reagents were removed, the enzyme was treated with methyl(14C)acetimidate. Enzyme activity was enhanced 13-fold as 1.1 (14C)acetimidyl group was incorporated per active site. A labeled peptide was isolated from a tryptic-chymotryptic digest of the modified enzyme in 35% overall yield. Amino acid composition and sequential Edman degradations identified the peptide as residues 219-229; lysine residue 228 was modified with the radioactive acetimidyl group.

Veronese FM, Degani Y, Nyc JF, Smith EL
Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. III. Isolation and sequences of peptides containing selectively labeled lysine and cysteine residues.
J Biol Chem. 1974; 249: 7936-41

DE VILLENEUVE VH, WADMAN SK, VAN DE HEUVEL JM
[A CHILD WITH A SPECIAL FORM OF PORPHYRIA].
Maandschr Kindergeneeskd. 1964; 32: 731-42

TOBIAS G
CONGENITAL PORPHYRIA IN A CAT.
J Am Vet Med Assoc. 1964; 145: 462-3

JORGENSEN SK, WITH TK
Porphyria in domestic animals: Danish observations in pigs and cattle and comparison with human porphyria.
Ann N Y Acad Sci. 1963; 104: 701-9

KOZLOWSKI B
[On the problem of late cutaneous porphyria].
Pol Tyg Lek. 1962; 17: 641-4

CHATTERJI AK, CHATTERJEA JB
Porphyria erythropoietica. Review of Indian literature and report of two new cases.
J Indian Med Assoc. 1962; 39: 526-30

SAVELLI G, CENCIOTTI L
[Rapid biochemical diagnosis of idiopathic porphyria].
Pathologica. 1961; 53: 333-5

ROSE JA, HELLMAN ES, TSCHUDY DP
Effect of diet on induction of experimental porphyria.
Metabolism. 1961; 10: 514-21

JANOUSEK V, OCENASEK M
[Excretion of delta-aminolevulinic acid and porphobilinogen in experimental porphyria in rabbits].
Cesk Fysiol. 1960; 9: 82-3

CURNOW DH, MORGAN EH, SARFATY GA
Acute intermittent porphyria. A family study.
Australas Ann Med. 1959; 8: 267-71

BARAC G, LECOMTE J, RAMIOUL H, STAINIER J
[Practical use of examining all members of a family for porphyria in a case of acute diagnosed porphyria in one of them].
Rev Med Liege. 1958; 13: 314-7

GELFAND M, MITCHELL JD
Bantu porphyria; a clinical study of sixteen cases.
Trans R Soc Trop Med Hyg. 1957; 51: 62-8

PFANDLER U
[Heterozygotic and homozygotic manifestation of certain metabolic disorders: chronic porphyria, cystinosis, Niemann-Pick disease].
Acta Genet Stat Med. 1957; 7: 184-7

LASSERRE R
[Porphyria: a common disease].
Rev Med Suisse Romande. 1957; 77: 919-28

MAUZERALL D, GRANICK S
The occurrence and determination of delta-amino-levulinic acid and porphobilinogen in urine.
J Biol Chem. 1956; 219: 435-46

BERGLUND H, KORNERUP T, PAUL KG
The treatment of photophobia in two cases of cutaneous porphyria.
Acta Ophthalmol (Copenh). 1955; 33: 247-51

SCHMID R, SCHWARTZ S, SUNDBERG RD
Erythropoietic (congenital) porphyria: A rare abnormality of the normoblasts.
Blood. 1955; 10: 416-28

DEROT M, CANIVET J
[A new case of cutaneous porphyria of adults].
Presse Med. 1955; 63: 97-8

SHEMIN D, RUSSELL CS, ABRAMSKY T
The succinate-glycine cycle. I. The mechanism of pyrrole synthesis.
J Biol Chem. 1955; 215: 613-26

GRANICK S
Enzymatic conversion of delta-amino levulinic acid to porphobilinogen.
Science. 1954; 120: 1105-6
Display abstract
An enzyme has been found in extracts of chicken erythrocytes which converts delta-amino levulinic acid to the monopyrrole porphobilinogen. Some of its properties have been described (6).

OTTOLENGHI-LODIGIANI F
[Congenital porphyria. III. Iron metabolism in congenital porphyria].
Soc Ital Dermatol Sifilogr Sezioni Interprov Soc Ital Dermatol Sifilogr. 1954; 95: 450-77

Bogorad L, Granick S
The Enzymatic Synthesis of Porphyrins from Porphobilinogen.
Proc Natl Acad Sci U S A. 1953; 39: 1176-88

PATON J, MANDER S, SHEPPARD R, EY G
A case of brucellosis with acute porphyria.
Med J Aust. 1952; 1: 781-4

OTTOLENGHI-LODIGIANI F, SERCHI G
[Porphyrin and its metabolism in congenital porphyria; studies on porphyrin in blood, in feces, in saliva, and in gastric and duodenal juices].
Soc Ital Dermatol Sifilogr Sezioni Interprov Soc Ital Dermatol Sifilogr. 1952; 93: 1-47

GRAY CH
Isotope studies in porphyria.
Br Med Bull. 1952; 8: 229-33

BILLION TJ Jr
Porphyria mixed--case report.
SD J Med Pharm. 1951; 4: 321-2

RIMINGTON C, MILES PA
A study of the porphyrins excreted in the urine by a case of congenital porphyria.
Biochem J. 1951; 50: 202-6

AVEZZU G
[Ascorbic acid epicatechin and pigmentogenesis of Pseudomonas aeruginosa].
Boll Soc Ital Biol Sper. 1951; 27: 105-7

TODD CM, GALLAGHER DJ
Acute porphyria with causalgic features; report of a case.
N Z Med J. 1951; 50: 480-5

RAMBERT P, CANIVET J, LAMY R
[Case of latent porphyria].
Bull Mem Soc Med Hop Paris. 1950; 66: 1405-13

BERLIN L, COTTON R
Gastro-intestinal manifestations of porphyria.
Am J Dig Dis. 1950; 17: 110-4

ALDRICH RA, HAWKINSON V, GRINSTEIN M, SCHWARTZ SO, WATSON CJ
Identification and crystallization of the porphyrins in the blood of a child with light-sensitive (congenital) porphyria.
Am J Dis Child. 1950; 79: 1125-6

FULLER RH
Acute porphyria; report of a case with post-mortem findings.
U S Armed Forces Med J. 1950; 1: 214-7

Dobriner K, Strain WH, Guild H, Localio SA
THE EXCRETION OF PORPHYRINS IN CONGENITAL PORPHYRIA.
J Clin Invest. 1938; 17: 761-4