NORMAL GENOMIC

• Dong C, Yang J, Leimkuhler S, Kirk ML
• Pyranopterin dithiolene distortions relevant to electron transfer in xanthine oxidase/dehydrogenase.
• Inorg Chem. 2014; 53: 7077-9
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• The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo(IV)-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.

• Sparacino-Watkins C, Stolz JF, Basu P
• Nitrate and periplasmic nitrate reductases.
• Chem Soc Rev. 2014; 43: 676-706
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• The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types--periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed.

• Strijkstra A et al.
• Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.
• Appl Environ Microbiol. 2014; 80: 7592-603
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• The betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene- versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. "A. aromaticum" pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of "Thauera" sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate beta-oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.

• Clark IC, Melnyk RA, Engelbrektson A, Coates JD
• Structure and evolution of chlorate reduction composite transposons.
• MBio. 2013; 4: 0-0
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• The genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algae ACDC) and three previously isolated strains (Ideonella dechloratans, Pseudomonas sp. strain PK, and Dechloromarinus chlorophilus NSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificans BC plasmid pALIDE01 and Pseudomonas chloritidismutans AW-1). De novo assembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualization. Phylogenetic analysis places chlorate reductase within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of enzymes with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding chlorite dismutase (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate that cld has been mobilized at least once from a perchlorate reducer to build chlorate respiration. IMPORTANCE: Genome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction.

• Brodkorb D, Gottschall M, Marmulla R, Luddeke F, Harder J
• Linalool dehydratase-isomerase, a bifunctional enzyme in the anaerobic degradation of monoterpenes.
• J Biol Chem. 2010; 285: 30436-42
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• Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme approximately 1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V(max)) were 140 nanokatals mg(-1) for the linalool dehydratase and 410 nanokatals mg(-1) for the geraniol isomerase, with apparent K(m) values of 750 mum and 500 mum, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).

• Cerqueira NM et al.
• The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase.
• J Comput Chem. 2009; 30: 2466-84
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• The catalytic mechanism of nitrate reduction by periplasmic nitrate reductases has been investigated using theoretical and computational means. We have found that the nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the Mo(V) species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with Mo(VI) ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of Mo(V) species. Mo(VI) intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the Mo(VI) species allow water molecule dissociation, and, the concomitant enzymatic turnover.

• Hofmann M
• Density functional theory study of model complexes for the revised nitrate reductase active site in Desulfovibrio desulfuricans NapA.
• J Biol Inorg Chem. 2009; 14: 1023-35
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• [Mo(SSCH3)(S2C2(CH3)2)2](x) complexes with charges x between -3 and +3 were investigated by density functional theory computations as minimal nitrate reductase active-site models. The strongly reduced species (x = -2, -3) exist preferentially as pentacoordinate sulfo complexes separated from a thiolate anion. The oxidized extremes (x > 0) clearly prefer hexacoordinate complexes with an eta(2)-MeSS ligand. Among the neutral and especially for the singly negatively charged species structures with eta(2)-MeSS and eta(1)-MeSS ligands are energetically close to the sulfo methyl sulfide complex without SS bonding. For x = -1 the three isomers lie in a 1.5 kcal mol(-1) energy range. Putative mechanistic pathways for nitrate reduction from the literature were investigated computationally: (1) reduction at a pentacoordinate sulfo complex, (2) reduction at the ligand, and (3) reduction at the molybdenum center with an R-S-S ligand. All three pathways could be traced at least for some overall charges but no definite conclusion can be drawn about the mechanism. Complexes with larger dithiolato ligands were also computed in order to model the tricyclic metallopterin framework more accurately: the first heterocyclus (5,6-dihydro-2H-pyran) stabilizes the nitrate complex and the molybdenum oxo product complex by approximately 10 kcal mol(-1) and also reduces the activation barrier (by approximately 5 kcal mol(-1)). The effect of the second (1,2,3,4-tetrahydropyrazin) and third heterocyclus (2-amino-3H-pyrimidin-4-one) on the relative energies is relatively small. For bigger models derived from an experimental protein structure, nitrate reduction at a persulfo molybdenum(IV) complex fragment (mechanism 3) is clearly favored over the oxidation of a molybdenum-bound sulfur atom (mechanism 2). Mechanism 1 could not be investigated for the big models but seems the least favorable on the basis of the results from smaller models.

• Carmona M et al.
• Anaerobic catabolism of aromatic compounds: a genetic and genomic view.
• Microbiol Mol Biol Rev. 2009; 73: 71-133
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• Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

• de Visser SP, Tahsini L, Nam W
• How does the axial ligand of cytochrome P450 biomimetics influence the regioselectivity of aliphatic versus aromatic hydroxylation?
• Chemistry. 2009; 15: 5577-87
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• The catalytic activity of high-valent iron-oxo active species of heme enzymes is known to be dependent on the nature of the axial ligand trans to the iron-oxo group. In a similar fashion, experimental studies on iron-oxo porphyrin biomimetic systems have shown a significant axial ligand effect on ethylbenzene hydroxylation, with an axial acetonitrile ligand leading to phenyl hydroxylation products and an axial chloride anion giving predominantly benzyl hydroxylation products. To elucidate the fundamental factors that distinguish this regioselectivity reversal in iron-oxo porphyrin catalysis, we have performed a series of density functional theory calculations on the hydroxylation of ethylbenzene by [Fe(IV)=O(Por(+.))L] (Por = porphyrin; L = NCCH(3) or Cl(-)), which affords 1-phenylethanol and p-ethylphenol products. The calculations confirm the experimentally determined product distributions. Furthermore, a detailed analysis of the electronic differences between the two oxidants shows that their reversed regioselectivity is a result of differences in orbital interactions between the axial ligand and iron-oxo porphyrin system. In particular, three high-lying orbitals (pi*(xz), pi*(yz) and a(2u)), which are singly occupied in the reactant complex, are stabilised with an anionic ligand such as Cl(-), which leads to enhanced HOMO-LUMO energy gaps. As a consequence, reactions leading to cationic intermediates through the two-electron reduction of the metal centre are disfavoured. The aliphatic hydroxylation mechanism, in contrast, is a radical process in which only one electron is transferred in the rate-determining transition state, which means that the effect of the axial ligand on this mechanism is much smaller.

• Diaz A, Valdes VJ, Rudino-Pinera E, Horjales E, Hansberg W
• Structure-function relationships in fungal large-subunit catalases.
• J Mol Biol. 2009; 386: 218-32
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• Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H(2)O(2)) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H(2)O(2) to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-gamma-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H(2)O(2) concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

• Tan X, Lindahl PA
• Tunnel mutagenesis and Ni-dependent reduction and methylation of the alpha subunit of acetyl coenzyme A synthase/carbon monoxide dehydrogenase.
• J Biol Inorg Chem. 2008; 13: 771-8
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• Two isolated alpha subunit mutants (A110C and A222L) of the alpha(2)beta(2) acetyl coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) from Moorella thermoacetica were designed to block the CO-migrating tunnel in the alpha subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated alpha subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the "majority" activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe(4)S(4) cubane in the apo-alpha subunit and the Ni-activated alpha subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-alpha subunit reduction was monophasic while Ni-activated alpha subunit reduction was biphasic, with the more rapid phase coincident with that of apo-alpha subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe(4)S(4)](2+) cubane. An upper limit of two electrons per alpha subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe(4)S(4)](2+) cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.

• Han D, Kim K, Oh J, Park J, Kim Y
• TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7.
• Proteins. 2008; 70: 900-14
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• Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.

• Gencic S, Grahame DA
• Two separate one-electron steps in the reductive activation of the A cluster in subunit beta of the ACDS complex in Methanosarcina thermophila.
• Biochemistry. 2008; 47: 5544-55
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• Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex found in methanogens and certain other Archaea that carries out the overall synthesis and cleavage of the acetyl C-C and C-S bonds of acetyl-CoA. The reaction is involved both in the autotrophic fixation of carbon and in the process of methanogenesis from acetate, and takes place at a unique active site metal center known as the A cluster, located on the beta subunit of the ACDS complex and composed of a binuclear Ni-Ni site bridged by a cysteine thiolate to an Fe4S4 center. In this work, a high rate of acetyl-CoA synthesis was achieved with the recombinant ACDS beta subunit by use of methylcobinamide as an appropriate mimic of the physiological base-off corrinoid substrate. The redox dependence of acetyl-CoA synthesis exhibited one-electron Nernst behavior, and the effects of pH on the observed midpoint potential indicated that reductive activation of the A cluster also involves protonation. Initial burst kinetic studies indicated the formation of stoichiometric amounts of an A cluster-acetyl adduct, further supported by direct chromatographic isolation of an active enzyme-acetyl species. Titration experiments indicated that two electrons are required for activation of the enzyme in the process of forming the enzyme-acetyl intermediate. The results also established that the A cluster-acetyl species undergoes reductive elimination of the acetyl group with the simultaneous release of two, low potential electron equivalents. Thus, the one-electron Nernst behavior can be interpreted as the sum of two separate, low potential, one-electron steps. The results tend to exclude reaction mechanisms involving either one- or three-electron reduced forms of the A cluster as immediate precursors to the acetyl species. A scheme involving a [Fe4S4]1+-Ni1+ species is favored over a [Fe4S4]2+-Ni0 form. The role of proton uptake in the possible formation of a Ni2+-hydride intermediate is also discussed. Trapping of electrons during the formation of the A cluster-acetyl species from substrates CO and methylcobinamide was found to be highly favorable, thus presenting a means for extensive activation of the enzyme under otherwise nonpermissive physiological redox potentials.

• Creevey NL, McEwan AG, Hanson GR, Bernhardt PV
• Thermodynamic characterization of the redox centers within dimethylsulfide dehydrogenase.
• Biochemistry. 2008; 47: 3770-6
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• Dimethylsulfide (DMS) dehydrogenase is a complex heterotrimeric enzyme that catalyzes the oxidation of DMS to DMSO and allows Rhodovulum sulfidophilum to grow under photolithotrophic conditions with DMS as the electron donor. The enzyme is a 164 kDa heterotrimer composed of an alpha-subunit that binds a bis(molybdopterin guanine dinucleotide)Mo cofactor, a polyferredoxin beta-subunit, and a gamma-subunit that contains a b-type heme. In this study, we describe the thermodynamic characterization of the redox centers within DMS dehydrogenase using EPR- and UV-visible-monitored potentiometry. Our results are compared with those of other bacterial Mo enzymes such as NarGHI nitrate reductase, selenate reductase, and ethylbenzene dehydrogenase. A remarkable similarity in the redox potentials of all Fe-S clusters is apparent.

• Pierce BS, Gardner JD, Bailey LJ, Brunold TC, Fox BG
• Characterization of the nitrosyl adduct of substrate-bound mouse cysteine dioxygenase by electron paramagnetic resonance: electronic structure of the active site and mechanistic implications.
• Biochemistry. 2007; 46: 8569-78
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• Mammalian cysteine dioxygenase (CDO) is a non-heme iron metalloenzyme that catalyzes the first committed step in oxidative cysteine catabolism. The active site coordination of CDO comprises a mononuclear iron ligated by the Nepsilon atoms of three protein-derived histidines, thus representing a new variant on the 2-histidine-1-carboxylate (2H1C) facial triad motif. Nitric oxide was used as a spectroscopic probe in investigating the order of substrate-O2 binding by EPR spectroscopy. In these experiments, CDO exhibits an ordered binding of l-cysteine prior to NO (and presumably O2) similar to that observed for the 2H1C class of non-heme iron enzymes. Moreover, the CDO active site is essentially unreactive toward NO in the absence of substrate, suggesting an obligate ordered binding of l-cysteine prior to NO. Typically, addition of NO to a mononuclear non-heme iron center results in the formation of an {FeNO}7 (S = 3/2) species characterized by an axial EPR spectrum with gx, gy, and gz values of approximately 4, approximately 4, and approximately 2, respectively. However, upon addition of NO to CDO in the presence of substrate l-cysteine, a low-spin {FeNO}7 (S = 1/2) signal that accounts for approximately 85% of the iron within the enzyme develops. Similar {FeNO}7 (S = 1/2) EPR signals have been observed for a variety of octahedral mononuclear iron-nitrosyl synthetic complexes; however, this type of iron-nitrosyl species is not commonly observed for non-heme iron enzymes. Substitution of l-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol did not produce any analogous {FeNO}7 signals (S = 1/2 or 3/2), thus reflecting the high substrate specificity of the enzyme observed by a number of researchers. The unusual {FeNO}7 (S = 1/2) electronic configuration adopted by the substrate-bound iron-nitrosyl CDO (termed {ES-NO}7) is a result of the bidentate thiol/amine coordination of l-cysteine in the NO-bound CDO active site. DFT computations were performed to further characterize this species. The DFT-predicted geometric parameters for {ES-NO}7 are in good agreement with the crystallographically determined substrate-bound active site configuration of CDO and are consistent with known iron-nitrosyl model complexes. Moreover, the computed EPR parameters (g and A values) are in excellent agreement with experimental results for this CDO species and those obtained from comparable synthetic {FeNO}7 (S = 1/2) iron-nitrosyl complexes.

• Szaleniec M, Hagel C, Menke M, Nowak P, Witko M, Heider J
• Kinetics and mechanism of oxygen-independent hydrocarbon hydroxylation by ethylbenzene dehydrogenase.
• Biochemistry. 2007; 46: 7637-46
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• Ethylbenzene dehydrogenase (EBDH) from the denitrifying bacterium Azoarcus sp. strain EbN1 (to be renamed Aromatoleum aromaticum) catalyzes the oxygen-independent, stereospecific hydroxylation of ethylbenzene to (S)-1-phenylethanol, the first known example of direct anaerobic oxidation of a nonactivated hydrocarbon. The enzyme is a trimeric molybdenum/iron-sulfur/heme protein of 155 kDa that is quickly inactivated in air in its reduced state. Enzyme activity can be coupled to ferricenium tetrafluoroborate, providing a convenient way for kinetic measurements. EBDH exhibits activity with a wide range of ethylbenzene analogues, which were analyzed for their kinetic parameters, stoichiometry, and formed products. The reactivity was correlated to the chemical structures by a quantitative structure-activity relationship (QSAR) model. On the basis of these results, quantum chemical calculations of DeltaG298 for formation of carbocations of the respective substrates were performed and used in reactivity analysis. A putative reaction mechanism is proposed on the basis of the experimental results and theoretical considerations. Finally, the enzyme reaction has been established in an electrochemical reactor, allowing sustained enzymatic reaction and potential technical applications of the enzyme.

• Zocher G, Winkler R, Hertweck C, Schulz GE
• Structure and action of the N-oxygenase AurF from Streptomyces thioluteus.
• J Mol Biol. 2007; 373: 65-74
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• Nitro groups are found in a number of bioactive compounds. Most of them arise by a stepwise mono-oxygenation of amino groups. One of the involved enzymes is AurF participating in the biosynthesis of aureothin. Its structure was established at 2.1 A resolution showing a homodimer with a binuclear manganese cluster. The enzyme preparation, which yielded the analyzed crystals, showed activity using in vitro and in vivo assays. Chain fold and cluster are homologous with ribonucleotide reductase subunit R2 and related enzymes. The two manganese ions and an iron content of about 15% were established by anomalous X-ray diffraction. A comparison of the cluster with more common di-iron clusters suggested an additional histidine in the coordination sphere to cause the preference for manganese over iron. There is no oxo-bridge. The substrate p-amino-benzoate was modeled into the active center. The model is supported by mutant activity measurements. It shows the geometry of the reaction and explains the established substrate spectrum.

• Zocher G, Wiesand U, Schulz GE
• High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli.
• FEBS J. 2007; 274: 5382-9
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• The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 A resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O-acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction.

• Shima S, Thauer RK
• A third type of hydrogenase catalyzing H2 activation.
• Chem Rec. 2007; 7: 37-46
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• The activation of molecular hydrogen is of interest both from a chemical and biological viewpoint. The covalent bond of H(2) is strong (436 kJ mol(-1)). Its cleavage is catalyzed by metals or metal complexes in chemical hydrogenation reactions and by metalloenzymes named hydrogenases in microorganisms. Until recently only two types of hydrogenases are known, the [FeFe[-hydrogenases and [NiFe[-hydrogenases. Both types, which are phylogenetically unrelated, harbor in their active site a dinuclear metal center with intrinsic CO and cyanide ligands and contain iron-sulfur clusters for electron transport as revealed by their crystal structures. Fifteen years ago a third type of phylogenetically unrelated hydrogenase was discovered, which has a mononuclear iron active site and is devoid of iron-sulfur clusters. It was initially referred to as "metal free" hydrogenase, but was later renamed iron-sulfur cluster-free hydrogenase or [Fe[-hydrogenase. In this review, we introduce first the [FeFe[-hydrogenases and [NiFe[-hydrogenases, and then focus on the structure and function of the iron-sulfur cluster-free hydrogenase (Hmd) and show that this enzyme contains an iron-containing cofactor. The low-spin iron is complexed by two intrinsic CO-, one sulfur- and one or two N/O ligands and has one open coordination site, which is proposed to be the location of H(2) binding.

• Marco-Marin C, Gil-Ortiz F, Perez-Arellano I, Cervera J, Fita I, Rubio V
• A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase.
• J Mol Biol. 2007; 367: 1431-46
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• Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 A and 2.5 A resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha(3)beta(8)alpha(4) sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the beta sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta(5)beta(4) sandwich fold and three alpha helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate.

• Botar B, Kogerler P, Hill CL
• A nanoring-nanosphere molecule, {Mo214V30}: pushing the boundaries of controllable inorganic structural organization at the molecular level.
• J Am Chem Soc. 2006; 128: 5336-7
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• A controlled, Raman-monitored chemical reduction of a molybdate and vanadate mixture affords a new type of molybdenum-oxide-based cluster showing an unprecedented level of inorganic structural organization. The cluster incorporates two nanosized substructures (a ring and a sphere) in an open clam-like assembly. Multiple methods indicate that the nanoring contains delocalized electrons and the nanosphere contains localized but interacting electrons.

• Hiromoto T, Fujiwara S, Hosokawa K, Yamaguchi H
• Crystal structure of 3-hydroxybenzoate hydroxylase from Comamonas testosteroni has a large tunnel for substrate and oxygen access to the active site.
• J Mol Biol. 2006; 364: 878-96
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• The 3-hydroxybenzoate hydroxylase (MHBH) from Comamonas testosteroni KH122-3s is a single-component flavoprotein monooxygenase, a member of the glutathione reductase (GR) family. It catalyzes the conversion of 3-hydroxybenzoate to 3,4-dihydroxybenzoate with concomitant requirements for equimolar amounts of NADPH and molecular oxygen. The production of dihydroxy-benzenoid derivative by hydroxylation is the first step in the aerobic degradation of various phenolic compounds in soil microorganisms. To establish the structural basis for substrate recognition, the crystal structure of MHBH in complex with its substrate was determined at 1.8 A resolution. The enzyme is shown to form a physiologically active homodimer with crystallographic 2-fold symmetry, in which each subunit consists of the first two domains comprising an active site and the C-terminal domain involved in oligomerization. The protein fold of the catalytic domains and the active-site architecture, including the FAD and substrate-binding sites, are similar to those of 4-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PHHY), which are members of the GR family, providing evidence that the flavoprotein aromatic hydroxylases share similar catalytic actions for hydroxylation of the respective substrates. Structural comparison of MHBH with the homologous enzymes suggested that a large tunnel connecting the substrate-binding pocket to the protein surface serves for substrate transport in this enzyme. The internal space of the large tunnel is distinctly divided into hydrophilic and hydrophobic regions. The characteristically stratified environment in the tunnel interior and the size of the entrance would allow the enzyme to select its substrate by amphiphilic nature and molecular size. In addition, the structure of the Xe-derivative at 2.5 A resolution led to the identification of a putative oxygen-binding site adjacent to the substrate-binding pocket. The hydrophobic nature of the xenon-binding site extends to the solvent through the tunnel, suggesting that the tunnel could be involved in oxygen transport.

• Daruzzaman A, Clifton IJ, Adlington RM, Baldwin JE, Rutledge PJ
• Unexpected oxidation of a depsipeptide substrate analogue in crystalline isopenicillin N synthase.
• Chembiochem. 2006; 7: 351-8
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• Isopenicillin N synthase (IPNS) is a non-heme iron(ii)-dependent oxidase that is central to penicillin biosynthesis. Herein, we report mechanistic studies of the IPNS reaction in the crystalline state, using the substrate analogue delta-(L-alpha-aminoadipoyl)-(3R)-methyl-L-cysteine D-alpha-hydroxyisovaleryl ester (AmCOV) to probe the early stages of the catalytic cycle. The X-ray crystal structure of the anaerobic IPNS:Fe(II):AmCOV complex was solved to 1.40 A resolution, and it reveals several subtle differences in the active site relative to the complex of the enzyme with its natural substrate. The crystalline IPNS:Fe(II):AmCOV complex was then exposed to oxygen gas at high pressure; this brought about reaction to give what appears to be a hydroxymethyl/ene-thiol product. A mechanism for this reaction is proposed. These results offer further insight into the delicate interplay of steric and electronic effects in the IPNS active site and the mechanistic intricacies of this remarkable enzyme.

• Kappler U, Bailey S
• Molecular basis of intramolecular electron transfer in sulfite-oxidizing enzymes is revealed by high resolution structure of a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit.
• J Biol Chem. 2005; 280: 24999-5007
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• Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 A) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.

• Johnson HA, Pelletier DA, Spormann AM
• Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme.
• J Bacteriol. 2001; 183: 4536-42
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• The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

• RAVAL DN, WOLFE RG
• Malic dehydrogenase. II. Kinetic studies of the reaction mechanism.
• Biochemistry. 1962; 1: 263-9

• ROSE ZB
• Studies on the mechanism of action of isocitric dehydrogenase.
• J Biol Chem. 1960; 235: 928-33

• KING TE, HOWARD RL
• A soluble DPNH dehydrogenase from heart-muscle particles.
• Biochim Biophys Acta. 1960; 37: 557-9

• LANDON EJ, CARTER CE
• The preparation, properties, and inhibition of hypoxanthine dehydrogenase of avian kidney.
• J Biol Chem. 1960; 235: 819-24

• FRIEDEN C
• Glutamic dehydrogenase. III. The order of substrate addition in the enzymatic reaction.
• J Biol Chem. 1959; 234: 2891-6

• ENGLARD S, COLOWICK SP
• On the mechanism of an anaerobic exchange reaction catalyzed by succinic dehydrogenase preparations.
• Science. 1955; 121: 866-7