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.     

N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, an acyclic amide substrate of beta-lactamases: importance of the shape of the substrate in beta-lactamase evolution

Certain acyclic depsipeptides, but not peptides, are substrates of typical beta-lactamases [Pratt, R.F., & Govardhan, C.P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1302]. This may reflect either the greater chemical reactivity of depsipeptides (and of beta-lactams, the natural substrates) than peptides or the greater ease of distortion of the depsipeptide (ester) than the peptide (amide) group into a penicillin-like conformation. The latter explanation has been shown to be more likely by employment of a novel beta-lactamase substrate. N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, which combines a high chemical reactivity with a close to tetrahedral amide nitrogen atom. Although this substrate was better (higher kcat/KM) than a comparable depsipeptide for beta-lactamases, it was poorer than the depsipeptide for the Streptomyces R61 D-alanyl-D-alanine peptidase (which catalyzes specific peptide hydrolysis). It therefore seems likely that one vital feature of the putative evolution of a DD-peptidase into a beta-lactamase would have been modification of the active site to, on one hand, accommodate bicyclic beta-lactams and, on the other, exclude productive binding of planar acyclic amides. Certain serine beta-lactamases and the R61 DD-peptidase also catalyze methanolysis and aminolysis by D-phenylalanine of the N-acylaziridine. The latter reaction, the first amide aminolysis shown to be catalyzed by a beta-lactamase, is a very close analogue of the transpeptidase reaction of DD-peptidases. The methanolysis reaction appeared to proceed by way of the same acyl-enzyme intermediate as formed from depsipeptides possessing the same acyl moiety as the aziridine. The kinetics of methanolysis were employed to determine whether acylation or deacylation was rate limiting to the hydrolysis reaction under saturating substrate concentrations. The kinetics of the aminolysis reaction, catalyzed by the Enterobacter cloacae P99 beta-lactamase, showed the characteristics of, and were interpreted in terms of, a sequential mechanism previously deduced for depsipeptides and this enzyme [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry 28, 6875-6882]. This mechanism features two separate binding sites, only one of which is productive. Strikingly, the binding of the N-acylaziridine to the nonproductive site was very tight, such that essentially all hydrolysis at substrate concentrations above 0.1Km proceeded via the ternary complex; this could also be true of penicillins.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes.

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria.     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.     

Inhibition of class D beta-lactamases by diaroyl phosphates

The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.     

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.     

Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams.

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.     

Streptomyces K15 DD-peptidase-catalysed reactions with ester and amide carbonyl donors.

In water, the purified 26 000-Mr membrane-bound DD-peptidase of Streptomyces K15 hydrolyses the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate (release of D-lactate) and the amide carbonyl donor Ac2-L-Lys-D-Ala-D-Ala (release of D-alanine) with accumulation of acyl- (Ac2-L-Lys-D-alanyl-)enzyme. Whereas hydrolysis of the ester substrate proceeds to completion, hydrolysis of the amide substrate is negligible because of the capacity of the K15 DD-peptidase for utilizing the released D-alanine in a transfer reaction (Ac2-L-Lys-D-Ala-D-Ala + D-Ala----Ac2-L-Lys-D-Ala-D-Ala + D-Ala) that maintains the concentration of the amide substrate at a constant level. In the presence of an amino acceptor X-NH2 (Gly-Gly or Gly-L-Ala) related to the Streptomyces peptidoglycan, both amide and ester carbonyl donors are processed without detectable accumulation of acyl-enzyme. Under proper conditions, the acceptor activity of water and, in the case of the amide substrate, the acceptor activity of the released D-alanine can be totally overcome so that the two substrates are quantitatively converted into transpeptidated product Ac2-L-Lys-D-Ala-NH-X (and hydrolysis is prevented). Experimental evidence suggests that the amino acceptor modifies both the binding of the carbonyl donor to the enzyme and the ensuing rate of enzyme acylation.     

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.     

Toward better antibiotics: crystallographic studies of a novel class of DD-peptidase/beta-lactamase inhibitors

Beta-lactam antibiotics are vital weapons in the treatment of bacterial infections, but their future is under increasing threat from beta-lactamases. These bacterial enzymes hydrolyze and inactivate beta-lactam antibiotics, rendering the host cell resistant to the bactericidal effects of the drugs. Nevertheless, the bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases), the killing targets of beta-lactams, remain attractive targets for antibiotic compounds. Cyclic acyl phosph(on)ates have been developed and investigated as potential inhibitors of both transpeptidases and beta-lactamases. The X-ray crystal structures of the complexes of the Streptomyces strain R61 DD-peptidase inhibited by a bicyclic [1-hydroxy-4,5-benzo-2,6-dioxaphosphorinanone(3)-1-oxide] and a monocyclic [1-hydroxy-4-phenyl-2,6-dioxaphosphorinanone(3)-1-oxide] acyl phosphate were determined to investigate the mode of action of these novel inhibitors. The structures show, first, that these inhibitors form covalent bonds with the active site serine residue of the enzyme and that the refractory complexes thus formed are phosphoryl-enzyme species rather than acyl enzymes. The complexes are long-lived largely because, after ring opening, the ligands adopt conformations that cannot directly recyclize, the latter a phenomenon previously observed with cyclic acyl phosph(on)ates. While the two inhibitors bind in nearly identical conformations, the phosphoryl-enzyme complex formed from the monocyclic compound is significantly less mobile than that formed from the bicyclic compound. Despite this difference, the complex with the bicyclic compound breaks down to regenerate free enzyme somewhat more slowly than that of the monocyclic. This may be because of steric problems associated with the reorientation of the larger bicyclic ligand required for reactivation. The structures are strikingly different in the orientation of the phosphoryl moiety from those generated using more specific phosph(on)ates. Models of the noncovalent complexes of the monocyclic compound with the R61 DD-peptidase and a structurally very similar class C beta-lactamase suggest reasons why the former enzyme is phosphorylated by this compound, while the latter is acylated. Finally, this paper provides information that will help in the design of additional DD-peptidase inhibitors with the potential to serve as leads in the development of novel antibiotics.     

Dipeptide binding to the extended active site of the Streptomyces R61 D-alanyl-D-alanine-peptidase: the path to a specific substrate

Bacterial cell walls are cross-linked in the final step of biosynthesis by specific D-alanyl-D-alanine(DD)-peptidases/transpeptidases. The natural substrates of these enzymes should therefore be segments of peptidoglycan, but high specificity for such structures has yet to be demonstrated. The binding of dipeptides to the extended substrate binding site of the Streptomyces R61 DD-peptidase has been studied by means of a fluorescent beta-lactam probe. It was found that dipeptides of structure Gly-L-Xaa have affinity for a subsite adjacent to the beta-lactam binding site. Hydrophobic peptides such as Gly-L-Met and Gly-L-aminocaprylic acid had the greatest affinity for this site, with dissociation constants in each case of 0.19 mM. A combination of this motif with the C-terminal D-alanyl-D-alanine moiety required of a DD-peptidase substrate yielded a new substrate, glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine. Steady-state kinetic measurements established this compound as the most specific peptide substrate yet discovered for a DD-peptidase by at least 3 orders of magnitude (k(cat) = 69 s(-1), K(m) = 7.9 microM, k(cat)/K(m) = 8.7 x 10(6) s(-1) M(-1)); acylation was rate-determining at saturation. This substrate, presumably not coincidentally, contains the acyl donor and acceptor moieties, appropriately separated, of the Streptomyces peptidoglycan structure. This general method of approach should be of value in the search for specific substrates and inhibitors (antibiotics) of other DD-peptidases.     

The active centres in penicillin-sensitive enzymes

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site no. 1. Interaction between the N-14 substituent of the bound molecule and binding site no. 2 induces changes in binding site no. 1. In turn, the catalytic site thus created increases the chemical reactivity of the beta-lactam amide bond. As the beta-lactam ring opens and acylates an enzyme serine residue, the interaction between the thiazolidine (or dihydrothiazine) ring and binding site no. 3 stabilizes the acyl-enzyme complex. Enzyme regeneration slowly proceeds either by direct elimination of the penicilloyl moiety or via C-5-C-6 splitting of the bound metabolite. The fragment arising from thiazolidine yields free N-formyl-D-penicillamine while the enzyme-linked N-acylglycyl fragment is immediately attacked by an exogenous nucleophile correctly positioned on the acceptor site. Similarly, the enzyme action on L-X-D-Ala-D-Ala terminated peptides is mediated via a binding site no. 1 that combines with D-Ala-D-Ala, a binding site no. 2 that interacts with the side chain of the preceding L-residue, an inducible catalytic site and an acceptor site. Enzymes are known that form a transitory L-X-D-Ala-enzyme complex where the acyl group is ester-linked to the same serine residue as that involved in the formation of the penicilloyl-enzyme complex (Waxman et al., this symposium). Other enzymes, however, may function as catalyst templates. Depending on the enzymes, the independence of the beta-lactam and L-X-D-Ala-D-Ala active centres is more or less pronounced.     

Crystal structures of complexes between the R61 DD-peptidase and peptidoglycan-mimetic beta-lactams: a non-covalent complex with a "perfect penicillin"

The bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases) are the killing targets of beta-lactams, the most important clinical defense against bacterial infections. However, due to the constant development of antibiotic-resistance mechanisms by bacteria, there is an ever-present need for new, more effective antimicrobial drugs. While enormous numbers of beta-lactam compounds have been tested for antibiotic activity in over 50 years of research, the success of a beta-lactam structure in terms of antibiotic activity remains unpredictable. Tipper and Strominger suggested long ago that beta-lactams inhibit DD-peptidases because they mimic the D-alanyl-D-alanine motif of the peptidoglycan substrate of these enzymes. They also predicted that beta-lactams having a peptidoglycan-mimetic side-chain might be better antibiotics than their non-specific counterparts, but decades of research have not provided any evidence for this. We have recently described two such novel beta-lactams. The first is a penicillin having the glycyl-L-alpha-amino-epsilon-pimelyl side-chain of Streptomyces strain R61 peptidoglycan, making it the "perfect penicillin" for this organism. The other is a cephalosporin with the same side-chain. Here, we describe the X-ray crystal structures of the perfect penicillin in non-covalent and covalent complexes with the Streptomyces R61 DD-peptidase. The structure of the non-covalent enzyme-inhibitor complex is the first such complex to be trapped crystallographically with a DD-peptidase. In addition, the covalent complex of the peptidyl-cephalosporin with the R61 DD-peptidase is described. Finally, two covalent complexes with the traditional beta-lactams benzylpenicillin and cephalosporin C were determined for comparison with the peptidyl beta-lactams. These structures, together with relevant kinetics data, support Tipper and Strominger's assertion that peptidoglycan-mimetic side-chains should improve beta-lactams as inhibitors of DD-peptidases.     

Mechanistic insights into the inhibition of serine proteases by monocyclic lactams.

Although originally discovered as inhibitors of pencillin-binding proteins, beta-lactams have more recently found utility as serine protease inhibitors. Indeed through their ability to react irreversibly with nucleophilic serine residues they have proved extraordinarily successful as enzyme inhibitors. Consequently there has been much speculation as to the reason for the general effectiveness of beta-lactams as antibacterials or inhibitors of hydrolytic enzymes. The interaction of analogous beta- and gamma-lactams with a serine protease was investigated. Three series of gamma-lactams based upon monocyclic beta-lactam inhibitors of elastase [Firestone, R. A. et al. (1990) Tetrahedron 46, 2255-2262.] but with an extra methylene group inserted between three of the bonds in the ring were synthesized. Their interaction with porcine pancreatic elastase and their efficacy as inhibitors were evaluated through the use of kinetic, NMR, mass spectrometric, and X-ray crystallographic analyses. The first series, with the methylene group inserted between C-3 and C-4 of the beta-lactam template, were readily hydrolyzed but were inactive or very weakly active as inhibitors. The second series, with the methylene group between C-4 and the nitrogen of the beta-lactam template, were inhibitory and reacted reversibly with PPE to form acyl-enzyme complexes, which were stable with respect to hydrolysis. The third series, with the methylene group inserted between C-2 and C-3, were not hydrolyzed and were not inhibitors consistent with lack of binding to PPE. Comparison of the crystal structure of the acyl-enzyme complex formed between PPE and a second series gamma-lactam and that formed between PPE and a peptide [Wilmouth, R. C., et al. (1997) Nat. Struct. Biol. 4, 456-462.] reveals why the complexes formed with this series were resistant to hydrolysis and suggests ways in which stable acyl-enzyme complexes might be obtained from monocyclic gamma-lactam-based inhibitors.     

Understanding the acylation mechanisms of active-site serine penicillin-recognizing proteins: a molecular dynamics simulation study

Beta-lactam antibiotics inhibit enzymes involved in the last step of peptidoglycan synthesis. These enzymes, also identified as penicillin-binding proteins (PBPs), form a long-lived acyl-enzyme complex with beta-lactams. Antibiotic resistance is mainly due to the production of beta-lactamases, which are enzymes that hydrolyze the antibiotics and so prevent them reaching and inactivating their targets, and to mutations of the PBPs that decrease their affinity for the antibiotics. In this study, we present a theoretical study of several penicillin-recognizing proteins complexed with various beta-lactam antibiotics. Hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations have been performed to understand the role of several residues, and pK(a) calculations have also been done to determine their protonation state. We analyze the differences between the beta-lactamase TEM-1, the membrane-bound PBP2x of Streptococcus pneumoniae, and the soluble DD-transpeptidase of Streptomyces K15.     

Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A beta-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.     

Reactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design.