NORMAL GENOMIC

• Iyer S et al.
• Crystal structure of X-prolyl aminopeptidase from Caenorhabditis elegans: A cytosolic enzyme with a di-nuclear active site.
• FEBS Open Bio. 2015; 5: 292-302
• Display abstract

• Eukaryotic aminopeptidase P1 (APP1), also known as X-prolyl aminopeptidase (XPNPEP1) in human tissues, is a cytosolic exopeptidase that preferentially removes amino acids from the N-terminus of peptides possessing a penultimate N-terminal proline residue. The enzyme has an important role in the catabolism of proline containing peptides since peptide bonds adjacent to the imino acid proline are resistant to cleavage by most peptidases. We show that recombinant and catalytically active Caenorhabditis elegans APP-1 is a dimer that uses dinuclear zinc at the active site and, for the first time, we provide structural information for a eukaryotic APP-1 in complex with the inhibitor, apstatin. Our analysis reveals that C. elegans APP-1 shares similar mode of substrate binding and a common catalytic mechanism with other known X-prolyl aminopeptidases.

• Surucu HA, Aksoy N, Ozgoztas O, Sezen H, Yesilova Y, Turan E
• Prolidase activity in chronic plaque psoriasis patients.
• Postepy Dermatol Alergol. 2015; 32: 82-7
• Display abstract

• INTRODUCTION: Psoriasis is a chronic, inflammatory, T-cell-mediated and hyperproliferative skin disease characterized by erythematous, squamous, sharply circumscribed and infiltrated plaques. The metabolisms of the collagen proteins undergo considerable changes due to the acceleration of their turnovers as a result of increased prolidase activity in psoriasis patients. AIM: To determine the level of prolidase activity in psoriasis patients and evaluate its relationship with the oxidative system. MATERIAL AND METHODS: The serum prolidase enzyme activity, total antioxidant levels and total oxidant levels of 40 psoriasis patients and a control group including 47 healthy individuals were analyzed by using their serum samples, and their oxidative stress indices were calculated. RESULTS: The prolidase levels (p < 0.01), total oxidant levels (p < 0.01) and oxidative stress index levels (p < 0.001) of the patient group were higher than the corresponding parameters in the control group. The total antioxidant level was low (p < 0.01). Although a positive correlation was found between the prolidase and total antioxidant levels and the total oxidant level, no correlation was found between prolidase and the oxidative stress index. CONCLUSIONS: It has been determined that the activity of the prolidase enzyme increases due to the increased collage turnover in psoriasis patients. Increased serum oxidant levels and oxidative stress indices values may play a role in the pathogenesis of psoriasis.

• Weaver J, Watts T, Li P, Rye HS
• Structural basis of substrate selectivity of E. coli prolidase.
• PLoS One. 2014; 9: 111531-111531
• Display abstract

• Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 A resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.

• Li Y, Chooi YH, Sheng Y, Valentine JS, Tang Y
• Comparative characterization of fungal anthracenone and naphthacenedione biosynthetic pathways reveals an alpha-hydroxylation-dependent Claisen-like cyclization catalyzed by a dimanganese thioesterase.
• J Am Chem Soc. 2011; 133: 15773-85
• Display abstract

• The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel alpha-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the alpha-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-beta-lactamase-type thioesterase (MbetaL-TE). The bifunctional MbetaL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon alpha-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of alpha-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MbetaL-TE is a dimanganese enzyme and requires both Mn(2+) cations for the observed activities. The MbetaL-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an alpha/beta hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.

• Ragheb D, Bompiani K, Dalal S, Klemba M
• Evidence for catalytic roles for Plasmodium falciparum aminopeptidase P in the food vacuole and cytosol.
• J Biol Chem. 2009; 284: 24806-15
• Display abstract

• The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 degrees C in the pH range 5.0-7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.

• Mitra S, Bennett B, Holz RC
• Mutation of H63 and its catalytic affect on the methionine aminopeptidase from Escherichia coli.
• Biochim Biophys Acta. 2009; 1794: 137-43
• Display abstract

• In order to gain insight into the mechanistic role of a flexible exterior loop near the active site, made up of Y62, H63, G64, and Y65, that has been proposed to play an important role in substrate binding and recognition in the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H63A enzyme was prepared. Mutation of H63 to alanine does not affect the ability of the enzyme to bind divalent metal ions. The specific activity of H63A EcMetAP-I was determined using four different substrates of varying lengths, namely, l-Met-p-NA, MAS, MGMM and MSSHRWDW. For the smallest/shortest substrate (l-Met-p-NA) the specific activity decreased nearly seven fold but as the peptide length increased, the specific activity also increased and became comparable to WT EcMetAP-I. This decrease in specific activity is primarily due to a decrease in the observed k(cat) values, which decreases nearly sixty-fold for l-Met-p-NA while only a four-fold decrease is observed for the tri- and tetra-peptide substrates. Interestingly, no change in k(cat) was observed when the octa-peptide MSSHRWDW was used as a substrate. These data suggest that H63 affects the hydrolysis of small peptide substrates whereas large peptides can overcome the observed loss in binding energy, as predicted from K(m) values, by additional hydrophilic and hydrophobic interactions.

• Graham SC, Guss JM
• Complexes of mutants of Escherichia coli aminopeptidase P and the tripeptide substrate ValProLeu.
• Arch Biochem Biophys. 2008; 469: 200-8
• Display abstract

• Aminopeptidase P (APPro) is a manganese-containing enzyme that catalyses the hydrolysis of the N-terminal residue of a polypeptide if the second residue is proline. Structures of APPro mutants with reduced or negligible activity have been determined in complex with the tripeptide substrate ValProLeu. In the complex of Glu383Ala APPro with ValProLeu one of the two metal sites is only partly occupied, indicating an essential role for Glu383 in metal binding in the presence of substrate. His361Ala APPro clearly possesses residual activity as the ValProLeu substrate has been cleaved in the crystals; difference electron density consistent with bound ProLeu dipeptide and a disordered Val amino acid is present at the active site. Contrary to previous suggestions, the His243Ala mutant is capable of binding substrate. The structure of the His243Ala APPro complex with ValProLeu shows that the peptide interacts with one of the active-site metal atoms via its terminal amino group. The implications of these complexes for the roles of the respective residues in APPro catalysis are discussed.

• Nocek B, Mulligan R, Bargassa M, Collart F, Joachimiak A
• Crystal structure of aminopeptidase N from human pathogen Neisseria meningitidis.
• Proteins. 2008; 70: 273-9

• 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
• Display abstract

• 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.

• Graham SC et al.
• Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: insights into substrate recognition and the mechanism of catalysis.
• Biochemistry. 2006; 45: 964-75
• Display abstract

• Aminopeptidase P (APPro) is a manganese-dependent enzyme that cleaves the N-terminal amino acid from polypeptides where the second residue is proline. APPro shares a similar fold, substrate specificity, and catalytic mechanism with methionine aminopeptidase and prolidase. To investigate the roles of conserved residues at the active site, seven mutant forms of APPro were characterized kinetically and structurally. Mutation of individual metal ligands selectively abolished binding of either or both Mn(II) atoms at the active site, and none of these metal-ligand mutants had detectable catalytic activity. Mutation of the conserved active site residues His243 and His361 revealed that both are required for catalysis. We propose that His243 stabilizes substrate binding through an interaction with the carbonyl oxygen of the requisite proline residue of a substrate and that His361 stabilizes substrate binding and the gem-diol catalytic intermediate. Sequence, structural, and kinetic analyses reveal that His350, conserved in APPro and prolidase but not in methionine aminopeptidase, forms part of a hydrophobic binding pocket that gives APPro its proline specificity. Further, peptides in which the required proline residue is replaced by N-methylalanine or alanine are cleaved by APPro, but they are extremely poor substrates due to a loss of interactions between the prolidyl ring of the substrate and the hydrophobic proline-binding pocket.

• Nakajima Y et al.
• Unusual extra space at the active site and high activity for acetylated hydroxyproline of prolyl aminopeptidase from Serratia marcescens.
• J Bacteriol. 2006; 188: 1599-606
• Display abstract

• The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 angstroms resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-betaNA (4-acetyloxyproline beta-naphthylamide) was a better substrate than Pro-betaNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.

• Zheng Y, Roberts RJ, Kasif S, Guan C
• Characterization of two new aminopeptidases in Escherichia coli.
• J Bacteriol. 2005; 187: 3671-7
• Display abstract

• Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.

• Michalska K, Brzezinski K, Jaskolski M
• Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate.
• J Biol Chem. 2005; 280: 28484-91
• Display abstract

• The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-A resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (alphabeta)(2). The (alphabeta) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an alphabetabetaalpha-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII.Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr(179), the N-terminal residue of subunit beta liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates.

• Yoshimoto T et al.
• Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism.
• J Mol Biol. 2004; 337: 399-416
• Display abstract

• Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.

• Im YJ et al.
• The active site of a lon protease from Methanococcus jannaschii distinctly differs from the canonical catalytic Dyad of Lon proteases.
• J Biol Chem. 2004; 279: 53451-7
• Display abstract

• ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (Mj-Lon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-angstroms resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the beta4-alpha2 segment is an alpha-helix, whereas it is a beta-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pK(a) of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.

• Li JY et al.
• Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity.
• J Biol Chem. 2004; 279: 21128-34
• Display abstract

• Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.

• Graham SC, Maher MJ, Simmons WH, Freeman HC, Guss JM
• Structure of Escherichia coli aminopeptidase P in complex with the inhibitor apstatin.
• Acta Crystallogr D Biol Crystallogr. 2004; 60: 1770-9
• Display abstract

• Aminopeptidase P (APPro) is a metalloprotease whose active site includes a dinuclear manganese(II) cluster. The enzyme cleaves the N-terminal residue from a polypeptide when the second residue is proline. A complex of Escherichia coli APPro (EcAPPro) with an inhibitor, apstatin [N-(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl-L-prolyl-L-prolyl-L-alaninamide], has been crystallized. Apstatin binds to the active site of EcAPPro with its N-terminal amino group coordinated to one of the two Mn(II) atoms at the metal centre. The apstatin hydroxyl group replaces a hydroxide ion which bridges the two metal atoms in the native enzyme. The first proline residue of apstatin lies in a small hydrophobic cleft. The structure of the apstatin-EcAPPro complex has been refined at 2.3 A resolution with residuals R = 0.179 and R(free) = 0.204. The structure of the complex illustrates how apstatin inhibits APPro and suggests how substrates may bind to the enzyme, but the basis of the proline-specificity remains elusive.

• Jorgensen AT, Norrby PO, Liljefors T
• Investigation of the metal binding site in methionine aminopeptidase by density functional theory.
• J Comput Aided Mol Des. 2002; 16: 167-79
• Display abstract

• All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions. This was the case for both of the systems studied; one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E. coli methionine aminopeptidase type I (eMetAP- 1). Another important structural issue is the identity of the bridging oxygen, which is part of either a water molecule or a hydroxide ion. Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions. By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion.

• Thorell S, Schurmann M, Sprenger GA, Schneider G
• Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family.
• J Mol Biol. 2002; 319: 161-71
• Display abstract

• Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.

• Kumar A et al.
• Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design.
• Structure. 2002; 10: 357-67
• Display abstract

• Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

• McVey CE, Walsh MA, Dodson GG, Wilson KS, Brannigan JA
• Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism.
• J Mol Biol. 2001; 313: 139-50
• Display abstract

• The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.

• Edwards RA, Whittaker MM, Whittaker JW, Baker EN, Jameson GB
• Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity.
• Biochemistry. 2001; 40: 4622-32
• Display abstract

• Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.

• Chiu CH, Lee CZ, Lin KS, Tam MF, Lin LY
• Amino acid residues involved in the functional integrity of Escherichia coli methionine aminopeptidase.
• J Bacteriol. 1999; 181: 4686-9
• Display abstract

• Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.

• van Asselt EJ, Dijkstra AJ, Kalk KH, Takacs B, Keck W, Dijkstra BW
• Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand.
• Structure. 1999; 7: 1167-80
• Display abstract

• BACKGROUND: Lytic transglycosylases are bacterial muramidases that catalyse the cleavage of the beta- 1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydrobond in the MurNAc residue. These muramidases play an important role in the metabolism of the bacterial cell wall and might therefore be potential targets for the rational design of antibacterial drugs. One of the lytic transglycosylases is Slt35, a naturally occurring soluble fragment of the outer membrane bound lytic transglycosylase B (MltB) from Escherichia coli. RESULTS: The crystal structure of Slt35 has been determined at 1.7 A resolution. The structure reveals an ellipsoid molecule with three domains called the alpha, beta and core domains. The core domain is sandwiched between the alpha and beta domains. Its fold resembles that of lysozyme, but it contains a single metal ion binding site in a helix-loop-helix module that is surprisingly similar to the eukaryotic EF-hand calcium-binding fold. Interestingly, the Slt35 EF-hand loop consists of 15 residues instead of the usual 12 residues. The only other prokaryotic proteins with an EF-hand motif identified so far are the D-galactose-binding proteins. Residues from the alpha and core domains form a deep groove where the substrate fragment GlcNAc can be bound. CONCLUSIONS: The three-domain structure of Slt35 is completely different from the Slt70 structure, the only other lytic transglycosylase of known structure. Nevertheless, the core domain of Slt35 closely resembles the fold of the catalytic domain of Slt70, despite the absence of any obvious sequence similarity. Residue Glu162 of Slt35 is in an equivalent position to Glu478, the catalytic acid/base of Slt70. GlcNAc binds close to Glu162 in the deep groove. Moreover, mutation of Glu162 into a glutamine residue yielded a completely inactive enzyme. These observations indicate the location of the active site and strongly support a catalytic role for Glu162.

• Strater N, Sherratt DJ, Colloms SD
• X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination.
• EMBO J. 1999; 18: 4513-22
• Display abstract

• The structure of aminopeptidase A (PepA), which functions as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 A resolution. In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular. In contrast, PepA homologues from other species have no known DNA-binding activity and are not implicated in transcriptional regulation or control of site-specific recombination. PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP). However, the N-terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19 degrees compared with its position in LAP. PepA is a homohexamer of 32 symmetry. A groove that runs from one trimer face across the 2-fold molecular axis to the other trimer face is proposed to be the DNA-binding site. Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3-fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right-handed superhelix such that three negative supercoils are trapped.

• Scolnick LR, Kanyo ZF, Cavalli RC, Ash DE, Christianson DW
• Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function.
• Biochemistry. 1997; 36: 10558-65
• Display abstract

• Arginase is a thermostable (Tm = 75 degrees C) binuclear manganese metalloenzyme which hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn2+A) and its ligand H101 in catalysis and thermostability. We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere [Cavalli, R. C., Burke, C. J., Kawamoto, S., Soprano, D. R., and Ash, D. E. (1994) Biochemistry 33, 10652-10657]. We conclude that the substitution of a wild-type histidine ligand to Mn2+A compromises metal binding, which in turn compromises protein thermostability and catalytic function. Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.

• Takusagawa F, Kamitori S, Markham GD
• Structure and function of S-adenosylmethionine synthetase: crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 28 angstroms resolution.
• Biochemistry. 1996; 35: 2586-96
• Display abstract

• S-Adenosylmethionine synthetase (MAT,ATP:L-methionine S-adenosltransferase, EC 2.5.1.6) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi), and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. The first crystal structure of MAT from Escherichia coli has recently been determined [Takusagawa et al. (1995) J. Biol. Chem. 271, 136-147]. In order to elucidate the active site and possible catalytic reaction mechanism, the MAT structures in the crystals grown with the substrate ATP (and BrATP) and the product PPi have been determined (space group P6(2)22; unit cell a = b = 128.9 Angstroms, c= 139.8 Angstroms, resolution limit 2.8 Angstroms; R O.19; Rfree 0.26). The enzyme consists of four identical subunits; two subunits form a spherical dimer, and pairs of these tightly bound dimers form a tetrameric enzyme. Each dimer has two active sites which are located between the subunits. Each subunit consists of three domains related to each other by a pseudo 3-fold symmetry. The crystal structures showed that the ATP molecules were hydrolyzed to ADP and Pi by the enzyme. Those products were found at the active site along with the essential metal ions (K+ and Mg2+). This rather unexpected finding was first confirmed by the structure of the complex with PPi and later by an HPLC analysis. The enzyme hydrolyzed ATP to ADP and Pi in 72 h under the same conditions as the crystallization of the enzyme. In the active site, the diphosphate moiety of ADP and Pi interacts extensively with amino acid residues from the two subunits of the enzyme, whereas the adenine and ribose moieties have little interaction with the enzyme. The enzyme structure is little changed upon binding ADP. All amino acid residues involved in the active site are found to be conserved in the 14 reported sequences of MAT from a wide range of organisms. Thus the structure determined in this study can be utilized as a model for other members of the MAT family. On the basis of the crystal structures, the catalytic reaction mechanisms of AdoMet formation and hydrolysis of tripolyphosphate are proposed.

• Qiu X, Pohl E, Holmes RK, Hol WG
• High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor.
• Biochemistry. 1996; 35: 12292-302
• Display abstract

• The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 A resolution and in complex with Mn2+ at 2.2 A resolution. The structure of the flexible third domain could be determined at this high resolution. It appears to contain five antiparallel strands exhibiting a fold very similar to the SH3 domain. A superposition of 46 equivalent C alpha atoms of DtxR and alpha-spectrin SH3 resulted in an rms deviation of 3.0 A. The sequence identity is only 7%. This third domain of DtxR appears to have no interactions with the DNA binding domain nor with the metal binding domain of the repressor. Yet, flexibility in the region between the second and the third domain allows in principle significant conformational changes such as might occur upon DNA binding. The two metal binding sites in the second domain have been unraveled in considerable detail. Metal binding site 1 was well occupied in both the cobalt and manganese structures and showed a surprising sulfate ion as ligand. The sulfate was proven beyond doubt by the high peak at its position in a selenate versus sulfate difference Fourier. The presence of the intriguing sulfate ion at such a crucial position near the metal corepressor suggests the possibility that under physiological conditions phosphate may act as a "co-corepressor" for this class of metal-regulated DNA binding proteins in Corynebacteria, Mycobacteria, and related organisms. The second metal binding site is significantly different in these two DtxR structures. In the 2.0 A cobalt structure, the site is not occupied by a metal ion. In the 2.2 A manganese structure the site is well occupied, at approximately the same position as observed previously in cadmium DtxR. The ligands are Glu105, His106, the carbonyl oxygen of Cys102, and a water molecule. The reasons for differential occupancy of this site in different structures are intriguing and require further investigations.

• Mirwaldt C, Korndorfer I, Huber R
• The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 A resolution.
• J Mol Biol. 1995; 246: 227-39
• Display abstract

• The crystal structure of dihydrodipicolinate synthase from E. coli was determined by multiple isomorphous replacement methods. The structure was refined at a resolution of 2.5 A and the final R-factor is 19.6% for 32,190 reflections between 10.0 A and 2.5 A and F > 2 sigma (F). The crystallographic asymmetric unit contains two monomers related by approximate 2-fold symmetry. A tetramer with approximate 222 symmetry is built up by crystallographic symmetry. The tetramer is almost planar with no contacts between the subunits related by the non-crystallographic dyad. The active sites are accessible from a wide water-filled channel in the center of the tetramer. The dihydrodipicolinate synthase monomer is composed of two domains. Each polypeptide chain is folded into an 8-fold alpha/beta barrel and a C-terminal alpha-helical domain comprising residues 224 to 292. The fold is similar to that of N-acetylneuraminate lyase. The active site lysine 161 is located in the alpha/beta barrel and has access via two entrances from the C-terminal side of the barrel.

• Arfin SM et al.
• Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.
• Proc Natl Acad Sci U S A. 1995; 92: 7714-8
• Display abstract

• Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

• Mattevi A, Valentini G, Rizzi M, Speranza ML, Bolognesi M, Coda A
• Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition.
• Structure. 1995; 3: 729-41
• Display abstract

• BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation. RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme. CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.

• Bazan JF, Weaver LH, Roderick SL, Huber R, Matthews BW
• Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P, and creatinase share a common fold.
• Proc Natl Acad Sci U S A. 1994; 91: 2473-7
• Display abstract

• Amino acid sequence comparison suggests that the structure of Escherichia coli methionine aminopeptidase (EC 3.4.11.18) and the C-terminal domain of Pseudomonas putida creatinase (EC 3.5.3.3) are related. A detailed comparison of the three-dimensional folds of the two enzymes confirms this homology: with an approximately 260-residue chain segment, 218 C alpha atoms of the structures superimpose within 2.5 A; only 41 of these overlapping positions (i.e., 19%) feature identical amino acids in the two protein chains. Notwithstanding this striking correspondence in structure, methionine aminopeptidase binds and is stimulated by Co2+, while creatinase is not a metal-dependent enzyme. Searches of protein data banks using sequence and structure-based profiles reveal other enzymes, including aminopeptidase P (EC 3.4.11.9), prolidase (EC 3.4.13.9), and agropine synthase, that likely share the same "pita-bread" fold common to creatinase and methionine aminopeptidase.

• Waksman G, Krishna TS, Williams CH Jr, Kuriyan J
• Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution. Implications for a large conformational change during catalysis.
• J Mol Biol. 1994; 236: 800-16
• Display abstract

• The crystal structures of three forms of Escherichia coli thioredoxin reductase have been refined: the oxidized form of the wild-type enzyme at 2.1 A resolution, a variant containing a cysteine to serine mutation at the active site (Cys138Ser) at 2.0 A resolution, and a complex of this variant with nicotinamide adenine dinucleotide phosphate (NADP+) at 2.3 A resolution. The enzyme mechanism involves the transfer of reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to a disulfide bond in the enzyme, via a flavin adenine dinucleotide (FAD). Thioredoxin reductase contains FAD and NADPH binding domains that are structurally similar to the corresponding domains of the related enzyme glutathione reductase. The relative orientation of these domains is, however, very different in the two enzymes: when the FAD domains of thioredoxin and glutathione reductases are superimposed, the NADPH domain of one is rotated by 66 degrees with respect to the other. The observed binding mode of NADP+ in thioredoxin reductase is non-productive in that the nicotinamide ring is more than 17 A from the flavin ring system. While in glutathione reductase the redox active disulfide is located in the FAD domain, in thioredoxin reductase it is in the NADPH domain and is part of a four-residue sequence (Cys-Ala-Thr-Cys) that is close in structure to the corresponding region of thioredoxin (Cys-Gly-Pro-Cys), with a root-mean-square deviation of 0.22 A for atoms in the disulfide bonded ring. There are no significant conformational differences between the structure of the wild-type enzyme and that of the Cys138Ser mutant, except that a disulfide bond is not present in the latter. The disulfide bond is positioned productively in this conformation of the enzyme, i.e. it stacks against the flavin ring system in a position that would facilitate its reduction by the flavin. However, the cysteine residues are relatively inaccessible for interaction with the substrate, thioredoxin. These results suggest that thioredoxin reductase must undergo conformational changes during enzyme catalysis. All three structures reported here are for the same conformation of the enzyme and no direct evidence is available as yet for such conformational changes. The simplest possibility is that the NADPH domain rotates between the conformation observed here and an orientation similar to that seen in glutathione reductase. This would alternately place the nicotinamide ring and the disulfide bond near the flavin ring, and expose the cysteine residues for reaction with thioredoxin in the hypothetical conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

• Yamaguchi H et al.
• Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution.
• J Mol Biol. 1993; 229: 1083-100
• Display abstract

• Glutathione synthetase (gamma-L-glutamyl-L-cysteine: glycine ligase (ADP-forming) EC 6.3.2.3: GSHase) catalyzes the synthesis of glutathione from gamma-L-glutamyl-L-cysteine and Gly in the presence of ATP. The enzyme from Escherichia coli is a tetramer with four identical subunits of 316 amino acid residues. The crystal structure of the enzyme has been determined by isomorphous replacement and refined to a 2.0 A resolution. Two regions, Gly164 to Gly167 and Ile226 to Arg241, are invisible on the electron density map. The refined model of the subunit includes 296 amino acid residues and 107 solvent molecules. The crystallographic R-factor is 18.6% for 17.914 reflections with F > 3 sigma between 6.0 A and 2.0 A. The structure consists of three domains: the N-terminal, central, and C-terminal domains. In the tetrameric molecule, two subunits that are in close contact form a tight dimer, two tight dimers forming a tetramer with two solvent regions. The ATP molecule is located in the cleft between the central and C-terminal domains. The ATP binding site is surrounded by two sets of the structural motif that belong to those respective domains. Each motif consists of an anti-parallel beta-sheet and a glycine-rich loop.

• Ji X, Zhang P, Armstrong RN, Gilliland GL
• The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution.
• Biochemistry. 1992; 31: 10169-84
• Display abstract

• The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)

• Asano Y, Kato Y, Yamada A, Kondo K
• Structural similarity of D-aminopeptidase to carboxypeptidase DD and beta-lactamases.
• Biochemistry. 1992; 31: 2316-28
• Display abstract

• The gene for D-aminopeptidase (dap) has been isolated from the bacterium Ochrobactrum anthropi SCRC C1-38 [Asano, Y., Nakazawa, A., Kato, Y., & Kondo, K. (1989) J. Biol. Chem. 264, 14233-14239] and its nucleotide sequence determined. An expression plasmid pC138DP (4.5 kb) was constructed by placing the gene downstream of the lac promoter of pUC19. The amount of the enzyme in the cell-free extract of Escherichia coli JM109/pC138DP was elevated to 288,000 units/L of culture, which is about 3600-fold over that of O. anthropi SCRC C1-38. The enzyme comprised about 30% of the total extractable cellular protein. The gene consisted of an open reading frame of 1560 nucleotides which specifies a protein of Mr 57,257. The deduced amino acid sequence of the enzyme showed that it is related to carboxypeptidase DD, beta-lactamases, and penicillin-binding proteins. Seven mutants of the enzyme were generated by site-specific mutagenesis to explore the roles of the residues of interest, around the sequence Ser61-Xaa-Xaa-Lys64, where Xaa is any amino acid, since the identical sequences also appear in the penicillin-recognizing peptide hydrolases with Ser at the active sites. The mutant enzymes expressed in E. coli were purified to homogeneity and kinetically characterized. Replacements of the site at Ser61 and Lys64 yielded mutants showing significantly reduced Vmax values, while most of the Km values remained unchanged. Changes at Cys60, which is adjacent to the likely active center Ser61, to Ser and Gly resulted in the production of enzyme less sensitive to PCMB, with almost unaltered Vmax/Km values. The enzyme appears to be a serine peptidase rather than a thiol one. The inhibition by PCMB in the wild-type enzyme may have been caused by a formation of a mercaptide bond between Cys 60 and PCMB. Considering that D-aminopeptidase, carboxypeptidase DD (a penicillin-binding protein), and beta-lactamase have a common feature in recognizing peptides containing D-amino acid and that the former two catalyze transpeptidation reactions with substrates containing D-alanyl-D-alanine moieties, we propose that the enzyme is a new member of the "penicillin-recognizing enzymes". We showed that the enzyme is actually inhibited by beta-lactam compounds, such as 6-APA, 7-ACA, benzylpenicillin, and ampicillin, although they are not the substrate for the enzyme. The relationship between the primary structures and the reactions catalyzed by D-aminopeptidase and other serine hydrolases beta-lactamases and carboxypeptidase DD is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

• Higgins DG, Bleasby AJ, Fuchs R
• CLUSTAL V: improved software for multiple sequence alignment.
• Comput Appl Biosci. 1992; 8: 189-91
• Display abstract

• The CLUSTAL package of multiple sequence alignment programs has been completely rewritten and many new features added. The new software is a single program called CLUSTAL V, which is written in C and can be used on any machine with a standard C compiler. The main new features are the ability to store and reuse old alignments and the ability to calculate phylogenetic trees after alignment. The program is simple to use, completely menu driven and on-line help is provided.

• Hoeffken HW, Knof SH, Bartlett PA, Huber R, Moellering H, Schumacher G
• Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida.
• J Mol Biol. 1988; 204: 417-33
• Display abstract

• The three-dimensional crystal structure of creatine amidinohydrolase (creatinase EC 3.5.3.3) from Pseudomonas putida, a dimeric enzyme with a molecular weight of 97,000, has been determined by multiple isomorphous replacement, averaging over the local dyad and restrained crystallographic refinement at 1.9 A with a crystallographic R-value of 17.7%. The asymmetric unit contains a dimer. The two chemically identical subunits consist of 403 residues each. A subunit is built up of two domains, a small N-terminal and a larger C-terminal domain. The small domain has a central seven-stranded beta pleated sheet with short helices on the outside. The large domain forms a six-stranded antiparallel beta half-barrel with helices on the outside. The two domains are connected by a segment that links two helices. The binding site of the competitive inhibitor carbamoyl sarcosine, a close analog of the substrate creatine, is located in the center of the large domain and partly covered by the small domain of the other subunit. The carbamoyl group is tightly co-ordinated to a water molecule, which presumably represents the nucleophile involved in hydrolysis of creatine. A catalytic mechanism is proposed on the basis of this structure.

• Yoshimoto T, Matsubara F, Kawano E, Tsuru D
• Prolidase from bovine intestine: purification and characterization.
• J Biochem. 1983; 94: 1889-96
• Display abstract

• Prolidase [iminodipeptidase, EC 3.4.13.9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30 min at 30 degrees C and retained half of the activity after 15 min at 40 degrees C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116,000 by gel filtration on Sephadex G-150 and 56,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X = amino acid).

• Lin LN, Brandts JF
• Role of cis-trans isomerism of the peptide bond in protease specificity. Kinetic studies on small proline-containing peptides and on polyproline.
• Biochemistry. 1979; 18: 5037-42