Crystal structure of a deacylation-defective mutant of penicillin-binding protein 5 at 2.3-A resolution

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a d-alanine carboxypeptidase, cleaving the C-terminal d-alanine residue from cell wall peptides. Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with beta-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acyl-enzyme complex (t(12) approximately 9 min). A Gly-105 --> Asp mutation in PBP 5 markedly impairs this beta-lactamase activity (deacylation), with only minor effects on acylation, and promotes accumulation of a covalent complex with peptide substrates. To gain further insight into the catalytic mechanism of PBP 5, we determined the three-dimensional structure of the G105D mutant form of soluble PBP 5 (termed sPBP 5') at 2.3 A resolution. The structure is composed of two domains, a penicillin binding domain with a striking similarity to Class A beta-lactamases (TEM-1-like) and a domain of unknown function. In addition, the penicillin-binding domain contains an active site loop spatially equivalent to the Omega loop of beta-lactamases. In beta-lactamases, the Omega loop contains two amino acids involved in catalyzing deacylation. This similarity may explain the high beta-lactamase activity of wild-type PBP 5. Because of the low rate of deacylation of the G105D mutant, visualization of peptide substrates bound to the active site may be possible.     

A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae

Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.     

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.     

Kinetics of beta-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae. Involvement of acylation and deacylation in beta-lactam resistance.

Kinetic interactions of beta-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K(d) of 0.9 mm, and the acylation rate constant, k(2) of 180 s(-1), have been determined for the first time. The millimolar range K(d) implies that the beta-lactam fits to the active site pocket of the penicillin-sensitive PBP rather poorly, whereas the extremely fast k(2) value indicates that this step contributes most of the binding affinity of the beta-lactam. The values of K(d) (4 mm) and k(2) (0.56 s(-1)) were also determined for PBP2x(R). The combined value of k(2)/K(d), known as overall binding efficiency, for PBP2x(R) (137 m(-1) s(-1)) was over 1000-fold slower than that for PBP2x (200,000 m(-1) s(-1)), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2x(R) comes from the decreased ( approximately 300-fold) k(2). Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k(3)) for the third step of the interactions were determined to be 8 x 10(-6) s(-1) for penicilloyl-PBP2x and 5.7 x 10(-4) s(-1) for penicilloyl-PBP2x(R), corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2x(R). Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x(R) complex (k(3) = 3 x 10(-4) s(-1)) as compared with that of cefotaxime-PBP2x complex (3.5 x 10(-6) s(-1)). This is the first time that such a significant increase of k(3) values was found for a beta-lactam-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta-lactams.     

Kinetic and mechanistic studies of penicillin-binding protein 2x from Streptococcus pneumoniae.

High molecular weight penicillin-binding proteins (PBPs) are bifunctional enzymes that build bacterial cell walls from the glycopeptide lipid II [GlcNAc-MurNAc(L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala)-pyrophosphate-undecaprenol] by a process involving disaccharide polymerization and peptide cross-linking. The latter reaction involves acyl-transfer chemistry in which the penultimate (D)Ala first acylates the active-site serine, with release of the terminal (D)Ala, and is then transferred to the epsilon-amine of a Lys on a neighboring pentapeptide chain. These enzymes also catalyze hydrolysis of specific thioester substrates and acylation by beta-lactam antibiotics. In this paper, we explore these latter two reactions and report mechanistic experiments on the reaction of Streptococcus pneumoniae PBP 2x with N-benzoyl-(D)Ala-thioacetic acid [Bz-(D)Ala-(S)Gly] and penicillin G. For these experiments, we used PBP 2x, a soluble form of PBP 2x in which the transmembrane domain was deleted. The following results are significant: (1) pH dependencies for acylation of PBP 2x by penicillin G and Bz-(D)Ala-(S)Gly are identical, suggesting that the same ionizable residues are involved in both reactions and that these residues play the same catalytic role in the two processes. On the basis of these results, we propose a mechanistic model that is also consistent with recently published structural data [Gordon, E., et al. (2000) J. Mol. Biol. 299, 477-485]. (2) Pre-steady-state experiments for the PBP 2x-catalyzed hydrolysis of Bz-(D)Ala-(S)Gly at pH 6.5 indicate that k(c) is principally rate-limited by acylation with some contribution from deacylation. The contribution of these steps to rate limitation is pH-dependent, with acylation entirely rate-limiting at pH values less than 5.5 and deacylation principally rate-limiting above pH 8.5. (3) Results of solvent isotope effect and proton inventory experiments for acylation suggest a complex process that is at least partially rate-limited by chemistry with some involvement of changes in solvation and/or enzyme conformation. (4) Analysis of activation parameters suggests that during the acylation of PBP 2x by penicillin G the inherent chemical stability of penicillin's amide bond, as manifested in the enthalpy of activation, is offset by a favorable entropy term that reflects penicillin's rotationally constrained bicyclic system, which presumably allows a less energetically demanding entry into the transition state for acylation.     

Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams.     

Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for beta-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by beta-lactam antibiotics.     

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.     

Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.     

pH,inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure-function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five alpha- and epsilon-substituted L-Lys-D-Ala-D-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6-12.3, and the k(cat)/K(m) vs. pH profile for activity against Ac(2)-L-Lys-D-Ala-D-Ala was bell-shaped, with pK(a)s at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed DD-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A beta-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.     

Resistance to beta-lactam antibiotics and its mediation by the sensor domain of the transmembrane BlaR signaling pathway in Staphylococcus aureus

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.     

Restoration of susceptibility of methicillin-resistant Staphylococcus aureus to beta-lactam antibiotics by acidic pH: role of penicillin-binding protein PBP 2a

Methicillin-resistant Staphylococcus aureus (MRSA) is a global scourge, and treatment options are becoming limited. The MRSA phenotype reverts to that of beta-lactam-sensitive S. aureus when bacteria are grown at pH 5.0 in broth and, more importantly from a medical perspective (protracted, relapsing infections), after phagocytosis by macrophages, where the bacteria thrive in the acidic environment of phagolysosomes. The central factor for the MRSA phenotype is the function of the penicillin-binding protein (PBP) 2a, which maintains transpeptidase activity while being poorly inhibited by beta-lactams because of a closed conformation of its active site. We document herein by binding, acylation/deacylation kinetics, and circular dichroism spectroscopy with purified PBP 2a that at acidic pH (i) beta-lactams interact with PBP 2a more avidly; (ii) the non-covalent pre-acylation complex exhibits a lower dissociation constant and an increased rate of acyl-enzyme formation (first-order rate constant) without change in hydrolytic deacylation rate; and (iii) PBP 2a undergoes a conformational change in the presence of the antibiotic consistent with the opening of the active site from the closed conformation. These observations argue that PBP 2a most likely evolved for its physiological function at pH 7 or higher by adopting a closed conformation, which is not maintained at acidic pH. Although at the organism level the effect of acidic pH on other biological processes in MRSA could not be discounted, our report should provide the impetus for closer examination of the properties of PBP 2a at low pH and thereby identifying novel points of intervention in combating this problematic organism.     

Mechanism of inhibition of RTEM-2 beta-lactamase by cephamycins: relative importance of the 7 alpha-methoxy group and the 3' leaving group

Cefoxitin is a poor substrate of many beta-lactamases, including the RTEM-2 enzyme. Fisher and co-workers [Fisher, J., Belasco, J. G., Khosla, S., & Knowles, J. R. (1980) Biochemistry 19, 2895-2901] showed that the reaction between cefoxitin and RTEM-2 beta-lactamase yielded a moderately stable acyl-enzyme whose hydrolysis was rate-determining to turnover at saturation. The present work shows first that the covalently bound substrate in this acyl-enzyme has a 5-exo-methylene-1,3-thiazine structure, i.e., that the good (carbamoyloxy) 3' leaving group of cefoxitin has been eliminated in formation of the acyl-enzyme. Such an elimination has recently been shown in another case to yield an acyl-beta-lactamase inert to hydrolysis [Faraci, W. S., & Pratt, R. F. (1985) Biochemistry 24, 903-910]. Thus the cefoxitin molecule has two potential sources of beta-lactamase resistance, the 7 alpha-methoxy group and the good 3' leaving group. That the latter is important in the present example is shown by the fact that with analogous substrates where no elimination occurs at the enzyme active site, such as 3'-de(carbamoyloxy)cefoxitin and 3'-decarbamoylcefoxitin, no inert acyl-enzyme accumulates. An analysis of the relevant rate constants shows that the 7 alpha-methoxy group weakens noncovalent binding and slows down both acylation and deacylation rates, but with major effect in the acylation rate, while elimination of the 3' leaving group affects deacylation only.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance

Penicillin-binding proteins (PBPs), the primary targets for beta-lactam antibiotics, are periplasmic membrane-attached proteins responsible for the construction and maintenance of the bacterial cell wall. Bacteria have developed several mechanisms of resistance, one of which is the mutation of the target enzymes to reduce their affinity for beta-lactam antibiotics. Here, we describe the structure of PBP2x from Streptococcus pneumoniae determined to 2.4 A. In addition, we also describe the PBP2x structure in complex with cefuroxime, a therapeutically relevant antibiotic, at 2.8 A. Surprisingly, two antibiotic molecules are observed: one as a covalent complex with the active-site serine residue, and a second one between the C-terminal and the transpeptidase domains. The structure of PBP2x reveals an active site similar to those of the class A beta-lactamases, albeit with an absence of unambiguous deacylation machinery. The structure highlights a few amino acid residues, namely Thr338, Thr550 and Gln552, which are directly related to the resistance phenomenon.     

Inhibition of cathepsin G by 2-amino-3,1-benzoxazin-4-ones: kinetic investigations and docking studies

A series of benzoxazinones was used to investigate the interaction of human cathepsin G with acyl-enzyme inhibitors. With respect to the primary specificity of cathepsin G, inhibitors with hydrophobic or basic residues at position 2 were included in the study. Parameters of the enzyme acylation and deacylation were determined by slow-binding kinetics in the presence of a chromogenic substrate. For selected inhibitors, the time course of the enzyme-catalyzed conversion of the inhibitors was followed. This approach was suitable to elucidate a rate-determining deacylation step. Docking simulations of the noncovalent enzyme-inhibitor complexes were performed and several clusters were analyzed for each inhibitor. The amino acids of the active site that participate in the binding of the inhibitors were determined. The arrangements in several clusters of an inhibitor were not uniform with respect to the orientation by which the inhibitor was bound in the S(1) pocket. Docking of the basic piperazino derivatives 6 and 10 indicated an interaction with Glu 226 at the bottom of the S(1) specificity pocket. The (N-methyl)benzylamino derivative 1 showed the strongest acylation rate (k(on)=1200 M(-1) s(-1)), which was attributed to a high extent of pseudo-productive orientations of the noncovalent preassociation complex.     

Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp:Asn170Gln with benzylpenicillin and cephaloridine

The serine-beta-lactamases hydrolyze beta-lactam antibiotics in a reaction that proceeds via an acyl-enzyme intermediate. The double mutation, E166D:N170Q, of the class A enzyme from Staphylococcus aureus results in a protein incapable of deacylation. The crystal structure of this beta-lactamase, determined at 2.3 A resolution, shows that except for the mutation sites, the structure is very similar to that of the native protein. The crystal structures of two acyl-enzyme adducts, one with benzylpenicillin and the other with cephaloridine, have been determined at 1.76 and 1.86 A resolution, respectively. Both acyl-enzymes show similar key features, with the carbonyl carbon atom of the cleaved beta-lactam bond covalently bound to the side chain of the active site Ser70, and the carbonyl oxygen atom in an oxyanion hole. The thiadolizine ring of the cleaved penicillin is located in a slightly different position than the dihydrothiazine ring of cephaloridine. Consequently, the carboxylate moieties attached to the rings form different sets of interactions. The carboxylate group of benzylpenicillin interacts with the side chain of Gln237. The carboxylate group of cephaloridine is located between Arg244 and Lys234 side chains and also interacts with Ser235 hydroxyl group. The interactions of the cephaloridine resemble those seen in the structure of the acyl-enzyme of beta-lactamase from Escherichia coli with benzylpenicillin. The side chains attached to the cleaved beta-lactam rings of benzylpenicillin and cephaloridine are located in a similar position, which is different than the position observed in the E. coli benzylpenicillin acyl-enzyme complex. The three modes of binding do not show a trend that explains the preference for benzylpenicillin over cephaloridine in the class A beta-lactamases. Rather, the conformational variation arises because cleavage of the beta-lactam bond provides additional flexibility not available when the fused rings are intact. The structural information suggests that specificity is determined prior to the cleavage of the beta-lactam ring, when the rigid fused rings of benzylpenicillin and cephaloridine each form different interactions with the active site.     

Mutations in the active site of penicillin-binding protein PBP2x from Streptococcus pneumoniae. Role in the specificity for beta-lactam antibiotics.

Penicillin-binding protein 2x (PBP2x) isolated from clinical beta-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for beta-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from beta-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 --> Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 --> Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand beta3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand beta3 conflicts with the negative charge of the beta-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. The in vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.     

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.     

A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases

It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.     

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.