Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins

Penicillin-binding proteins (PBPs), the target enzymes of beta-lactam antibiotics such as penicillins and cephalosporins, catalyze the final peptidoglycan cross-linking step of bacterial cell-wall biosynthesis. beta-Lactams inhibit this reaction because they mimic the D-alanyl-D-alanine peptide precursors of cell-wall structure. Prior crystallographic studies have described the site of beta-lactam binding and inhibition, but they have failed to show the binding of D-Ala-D-Ala substrates. We present here the first high-resolution crystallographic structures of a PBP, D-Ala-D-Ala-peptidase of Streptomyces sp. strain R61, non-covalently complexed with a highly specific fragment (glycyl-L-alpha-amino-epsilon-pimelyl-D-Ala-D-Ala) of the cell-wall precursor in both enzyme-substrate and enzyme-product forms. The 1.9A resolution structure of the enzyme-substrate Henri-Michaelis complex was achieved by using inactivated enzyme, which was formed by cross-linking two catalytically important residues Tyr159 and Lys65. The second structure at 1.25A resolution of the uncross-linked, active form of the DD-peptidase shows the non-covalent binding of the two products of the carboxypeptidase reaction. The well-defined substrate-binding site in the two crystallographic structures shows a subsite that is complementary to a portion of the natural cell-wall substrate that varies among bacterial species. In addition, the structures show the displacement of 11 water molecules from the active site, the location of residues responsible for substrate binding, and clearly demonstrate the necessity of Lys65 and or Tyr159 for the acylation step with the donor peptide. Comparison of the complexed structures described here with the structures of other known PBPs suggests the design of species-targeted antibiotics as a counter-strategy towards beta-lactamase-elicited bacterial resistance.     

Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.     

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.     

Deacylation transition states of a bacterial DD-peptidase

Beta-lactam antibiotics restrict bacterial growth by inhibiting DD-peptidases. These enzymes catalyze the final transpeptidation step in bacterial cell wall biosynthesis. Although much structural information is now available for these enzymes, the mechanism of the actual transpeptidation reaction has not been studied in detail. The reaction is known to involve a double-displacement mechanism with an acyl-enzyme intermediate, which can be attacked by water, specific amino acids, peptides, and other acyl acceptors. We describe in this paper an investigation of acyl acceptor specificity and assess the need for general base catalysis in the deacylation transition state of the Streptomyces R61 DD-peptidase. We show, by the criterion of solvent deuterium kinetic isotope effect measurements and proton inventories, that the transition states of specific and nonspecific substrates are very similar, at least with respect to proton motion. The transition states for attack (tetrahedral intermediate formation) by d-amino acids and Gly-l-Xaa dipeptides do not include a general base catalyst, while such catalysis is essential for reaction with water and d-alpha-hydroxy acids. D-Alpha-hydroxy acids act as acyl acceptors for glycyl substrates but not for more specific d-alanyl substrates; hydroxy acids actually behave, more generally, as mixed inhibitors of the DD-peptidase. The structural and mechanistic bases of these observations are discussed; they should inform transition state analogue design.     

Kinetic solvent isotope effects on the deacylation of specific acyl-papains. Proton inventory studies on the papain-catalysed hydrolyses of specific ester substrates: analysis of possible transition state structures.

1. The hydrolyses of the p-nitrophenyl esters of N-benzyloxycarbonylglycine, alpha-N-benzyloxycarbonyl-L-lysine and N-methoxycarbonyl-L-phenylalanylglycine catalysed by papain (EC 3.4.22.2) have been studied in solvents having a variable composition of 2H2O and H2O. 2. kcat., which represents deacylation in the papain-catalysed hydrolysis of reactive esters, is some 2.3-fold less in 2H2O compared with H2O. The magnitude of kcat. has been determined as a function of the 2H atom fraction of the solvent. 3. Both linear and non-linear methods of least-square regression analysis have been applied to the data in order to obtain best-fit parameter values for several three-parameter models which express kcat. in terms of the 2H atom fraction of the solvent. These models represent some possible modes of restructuring of the active site protonic configuration consequent upon transition state formation. 4. The results of curve fitting reveal an essentially linear dependence of kcat. upon the 2H atom fraction, and it may therefore be concluded that the isotope effect originates from a single proton which is in the process of transfer in the transition state. 5. It is postulated on the basis of this and other evidence that the mobile proton is transferred from an attacking water molecule to the imidazole side chain of His-159 during tetrahedral intermediate formation. This has the effect of stabilizing the transition state and promoting catalysis. The role of His-159 in deacylation is therefore to provide general base catalysis. 6. Models that involve two or more protons, such as a two-proton relay system analogous to that proposed for the serine proteinases, or a multiproton 'medium' effect, are considered unlikely on the basis of the data reported in this paper. 7. A more detailed examination of possible transition state structures reveals that the only structure compatible with available experimental data and consistent with certain theoretical predictions is one in which the proton translocated in concern with reorganization of the heavy atom framework. In addition, the transition state vibrations of the mobile proton are strongly coupled to those of the heavy atoms. These properties of the transition state are also manifest in the transition state for the deacylation of serine proteinases.     

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.     

The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99

The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.     

Effects of substituents on the rates of deacylation of substituted benzoyl papains. Role of a carboxylate residue in the catalytic mechanism.

The effect of ring substituents on the rates of deacylation of 8 meta- and para-substituted benzoyl papains was evaluated. The rate constants were found to depend upon a single ionizing group of pKa = 4.2--4.3, and to decrease by a factor of approximately 2.2 when measured in 94% D2O/H2O. The rates of deacylation are increased greatly by electron-withdrawing groups on the benzene ring. The Hammett rho value is 2.74 +/- 0.32. A plot of the rate constants for deacylation of the benzoyl papains against the corresponding constants for substituted benzoyl chymotrypsins generates a straight line of slope 1.0. This result suggests a very similar distribution of charge on the benzoyl moiety in the transition state for the two enzymes, which is interpreted in terms of the net charge of the transition state for the deacylation of nonspecific acyl papains being equal to--1 with the general base catalyzed assistance to the attack of water on the acyl enzyme being provided by the negatively charged Asp-158 rather than by the neutral Asn-175-His-159 hydrogen bond network. This result together with a survey of literature data suggests that the role of Asp-158 in papain catalysis has been underestimated. The evidence advanced to date in support of the proposition that an imidazolium-159-cysteine-25 thiolate ion pair exists in native papain is evaluated and considered to be insufficient to decide the issue.     

Re‐examining the role of Lys67 in class C β‐lactamase catalysis

Lys67 is essential for the hydrolysis reaction mediated by class C β‐lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C β‐lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2‐ to 3‐fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C β‐lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61‐fold decrease in k_cat, the deacylation rate. We have determined the structure of the K67R mutant by X‐ray crystallography both in apo and transition state‐analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C β‐lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.     

The roles of residues Tyr150, Glu272, and His314 in class C β-lactamases

Serine β-lactamases contribute widely to the β-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many “natural” and “artificial” mutants of these different enzymes have been isolated. This paper is essentially focused on class C β-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism. © 1996 Wiley-Liss, Inc.     

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.     

Induction of positive cooperativity by amino acid replacements within the C-terminal domain of Penicillium chrysogenum ATP sulfurylase

ATP sulfurylase from Penicillium chrysogenum is an allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is 42% identical to the conserved core of adenosine 5'-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is believed to provide the binding site for the allosteric effector, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Replacement of Cys-509 with either Tyr or Ser destabilizes the R state, resulting in an enzyme that is intrinsically cooperative at pH 8 in the absence of PAPS. The kinetics of C509Y resemble those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S resemble those of the wild type enzyme in the presence of PAPS. It is likely that the negative charge on the Cys-509 side chain helps to stabilize the R state. Treatment of the enzyme with a low level of trypsin results in cleavage at Lys-527, a residue that lies in a region analogous to a PAPS motif-containing mobile loop of true APS kinase. Both mutant enzymes were cleaved more rapidly than the wild type enzyme, suggesting that movement of the mobile loop occurs during the R to T transition.     

Inhibition of serine amidohydrolases by complexes of vanadate with hydroxamic acids

Serine beta-lactamases are inhibited by phosphonate monoester monoanions. These compounds phosphonylate the active site serine hydroxyl group to form inert, covalent complexes. Since spontaneous hydrolysis of these phosphonates is generally quite slow, the beta-lactamase active site must have considerable affinity for the (presumably) pentacoordinated phosphonyl transfer transition state. Structural analogs of such a transition state might well therefore be effective and novel beta-lactamase inhibitors. Complexes of vanadate with hydroxamic acids may be able to achieve such a structure. Indeed, mixtures of these two components, but neither one alone, were found to inhibit a typical class C beta-lactamase. A Job plot of the inhibition by vanadate/benzohydroxamic acid mixtures indicated that the inhibitor was a 1:1 complex for which an inhibition constant of 4.2 microM could be calculated. A bacterial DD-peptidase, structurally similar to the beta-lactamase, was also inhibited (K(i) = 22 microM) by this complex. A similar rationale would suggest that other serine hydrolases might also be inhibited by these mixtures. In fact, chymotrypsin was inhibited by a complex of vanadate with benzohydroxamic acid (K(i) = 10 microM) and elastase by a complex with acetohydroxamic acid (K(i) = 90 microM).     

Structural basis for the β‐lactamase activity of EstU1, a family VIII carboxylesterase

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β‐lactam antibiotics as well as the ester bond of p‐nitrophenyl esters. EstU1 assumes a β‐lactamase‐like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β‐lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β‐lactam antibiotics. This result explains the weak β‐lactamase activity of EstU1 compared with class C β‐lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum. Proteins 2013; 81:2045–2051. © 2013 Wiley Periodicals, Inc.     

Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes

Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70. X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme. Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and [Tyr70----Phe]AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state. When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.     

Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.     

On the importance of a methyl group in beta-lactamase evolution: free energy profiles and molecular modeling.

beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

Mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: role of the nucleotide positioning loop

The aminoglycoside antibiotic resistance kinases (APHs) and the Ser/Thr/Tyr protein kinases share structural and functional homology but very little primary sequence conservation (<5%). A region of structural, but not amino acid sequence, homology is the nucleotide positioning loop (NPL) that closes down on the enzyme active site upon binding of ATP. This loop region has been implicated in facilitating phosphoryl transfer in protein kinases; however, there is no primary sequence conservation between APHs and protein kinases in the NPL. There is an invariant Ser residue in all APH NPL regions, however. This residue in APH(3')-IIIa (Ser27), an enzyme widespread in aminoglycoside-resistant Enterococci, Streptococci, and Staphylococci, directly interacts with the beta-phosphate of ATP through the Ser hydroxymethyl group and the amide hydrogen in the 3D structure of the enzyme. Mutagenesis of this residue to Ala and Pro supported a role for the Ser amide hydrogen in nucleotide capture and phosphoryl transfer. A molecular model of the proposed dissociative transition state, which is consistent with all of the available mechanistic data, suggested a role for the amide of the adjacent Met26 in phosphoryl transfer. Mutagenesis studies confirmed the importance of the amide hydrogen and suggest a mechanism where Ser27 anchors the ATP beta-phosphate facilitating bond breakage with the gamma-phosphate during formation of the metaphosphate-like transition, which is stabilized by interaction with the amide hydrogen of Met26. The APH NPL therefore acts as a lever, promoting phosphoryl transfer to the aminoglycoside substrate, with the biological outcome of clinically relevant antibiotic resistance.     

The short‐chain oxidoreductase Q9HYA2 from Pseudomonas aeruginosa PAO1 contains an atypical catalytic center

The characteristic oxidation or reduction reaction mechanisms of short‐chain oxidoreductase (SCOR) enzymes involve a highly conserved Asp‐Ser‐Tyr‐Lys catalytic tetrad. The SCOR enzyme Q9HYA2 from the pathogenic bacterium Pseudomonas aeruginosa was recognized to possess an atypical catalytic tetrad composed of Lys118‐Ser146‐Thr159‐Arg163. Orthologs of Q9HYA2 containing the unusual catalytic tetrad along with conserved substrate and cofactor recognition residues were identified in 27 additional species, the majority of which are bacterial pathogens. However, this atypical catalytic tetrad was not represented within the Protein Data Bank. The crystal structures of unligated and NADPH‐complexed Q9HYA2 were determined at 2.3 Å resolution. Structural alignment to a polyketide ketoreductase (KR), a typical SCOR, demonstrated that Q9HYA2's Lys118, Ser146, and Arg163 superimposed upon the KR's catalytic Asp114, Ser144, and Lys161, respectively. However, only the backbone of Q9HYA2's Thr159 overlapped KR's catalytic Tyr157. The Thr159 hydroxyl in apo Q9HYA2 is poorly positioned for participating in catalysis. In the Q9HYA2–NADPH complex, the Thr159 side chain was modeled in two alternate rotamers, one of which is positioned to interact with other members of the tetrad and the bound cofactor. A chloride ion is bound at the position normally occupied by the catalytic tyrosine hydroxyl. The putative active site of Q9HYA2 contains a chemical moiety at each catalytically important position of a typical SCOR enzyme. This is the first observation of a SCOR protein with this alternate catalytic center that includes threonine replacing the catalytic tyrosine and an ion replacing the hydroxyl moiety of the catalytic tyrosine.