The targets of β-lactam antibiotics are bacterial DD-peptidases that catalyze the final steps of peptidoglycan biosynthesis. Bacterial resistance to β-lactams is achieved by the production of β-lactamases, enzymes that catalyze β-lactam hydrolysis. Structural studies of both of these groups of enzymes, their substrates and of β-lactams have led to the conclusion that β-lactamases have evolved from a DD-peptidase ancestor. Thus, the active sites of DD-peptidases and serine β-lactamases are very similar. Why is it then that the active site of a serine β-lactamase can catalyze hydrolysis of a β-lactam while that of a DD-peptidase cannot? In view of the active site similarities, why was it necessary for β-lactamases to evolve at all? The aim of this review is to examine our current understanding of these issues in terms of the crystal structures of the relevant enzymes that are now available, rounding off the analysis with speculation where necessary.

Escherichia coli PBP5 (penicillin-binding protein 5) is a dd-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal d-alanyl-d-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the l-lysine of the original was replaced by l-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5.

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, d-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 104 M–1 s–1) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

The targets of β-lactam antibiotics are bacterial DD-peptidases (penicillin-binding proteins). β-Lactam SAR studies over many years have demonstrated the importance of a specifically placed negative charge, usually carboxylate, on these molecules. We show here that neutral analogues of classical β-lactam antibiotics are of comparable activity to the originals against β-lactam-resistant high molecular mass DD-peptidases of the B1 class, a group that includes PBP2a of methicillin-resistant Staphylococcus aureus. These neutral β-lactams may direct new development of antibiotics against certain penicillin-resistant bacteria. These molecules do have antibiotic activity against Gram-positive bacteria.Keywords: antibiotics; inhibition; neutral β-lactams; Penicillin-binding proteins;

The effectiveness of β-lactam antibiotics is greatly limited by the ability of bacteria to produce β-lactamases. These enzymes catalyze the hydrolysis of β-lactams and thus loss of their antibiotic activity. The search for inhibitors of β-lactamases began soon after β-lactams were introduced into medical practice and continues today. Some time ago, we introduced a new class of covalent serine β-lactamase inhibitors, the O-aryloxycarbonyl hydroxamates, that inactivated these enzymes by a unique mechanism in which the active site became cross-linked. We describe in this paper some new variants of this class of inhibitor. First, we investigated compounds in which more polar hydroxamates were incorporated. These were generally not more active than the original compounds against representative class A and class C β-lactamases, but one of them, 1-(benzoyl)-O-(phenoxycarbonyl)-3-hydroxyurea, was significantly more stable in solution, thus revealing a useful platform for further design. Second, we describe a series of O-(arylphosphoryl) hydroxamates that are also irreversible inactivators of class A and class C β-lactamases, by phosphorylation of the enzyme, as revealed by mass spectra. These compounds did not, however, cross-link the enzyme active site. A striking feature of their structure–activity profile was that hydroxamate remained the leaving group on enzyme phosphorylation rather than aryloxide, even though the aryloxide was intrinsically the better leaving group, as indicated by pKa values and demonstrated by the products of hydrolysis in free solution. Model building suggested that this phenomenon arises from the relative affinity of the enzyme active site components for the two leaving groups. The results obtained for both groups of inhibitors are important for further optimization of these inhibitors.

Bacterial dd-peptidases are the targets of the β-lactam antibiotics. The sharp increase in bacterial resistance toward these antibiotics in recent years has stimulated the search for non-β-lactam alternatives. The substrates of dd-peptidases are elements of peptidoglycan from bacterial cell walls. Attempts to base dd-peptidase inhibitor design on peptidoglycan structure, however, have not been particularly successful to date because the specific substrates for most of these enzymes are unknown. It is known, however, that the preferred substrates of low-molecular mass (LMM) class B and C dd-peptidases contain the free N-terminus of the relevant peptidoglycan. Two very similar LMMC enzymes, for example, the Actinomadura R39 dd-peptidase and Bacillus subtilis PBP4a, recognize a d-α-aminopimelyl terminus. The peptidoglycan of B. subtilis in the vegetative stage, however, has the N-terminal d-α-aminopimelyl carboxylic acid amidated. The question is, therefore, whether the dd-peptidases of B. subtilis are separately specific to carboxylate or carboxamide or have dual specificity. This paper describes an investigation of this issue with B. subtilis PBP4a. This enzyme was indeed found to have a dual specificity for peptide substrates, both in the acyl donor and in the acyl acceptor sites. In contrast, the R39 dd-peptidase, from an organism in which the peptidoglycan is not amidated, has a strong preference for a terminal carboxylate. It was also found that acyl acceptors, reacting with acyl–enzyme intermediates, were preferentially d-amino acid amides for PBP4a and the corresponding amino acids for the R39 dd-peptidase. Examination of the relevant crystal structures, aided by molecular modeling, suggested that the expansion of specificity in PBP4a accompanies a change of Arg351 in the R39 enzyme and most LMMC dd-peptidases to histidine in PBP4a and its orthologs in other Bacillus sp. This histidine, in neutral form at pH 7, appeared to be able to favorably interact with both carboxylate and carboxamide termini of substrates, in agreement with the kinetic data. It may still be possible, in specific cases, to combat bacteria with new antibiotics based on particular elements of their peptidoglycan structure.

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to β-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-α-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics.

β-Lactamase inhibitors are important in medicine in the protection of β-lactam antibiotics from β-lactamase-catalyzed destruction. The most effective inhibitors of serine β-lactamases covalently modify the enzyme active site. We have recently studied O-acyl and O-phosphyl hydroxamates as a new class of such inhibitors. In this paper, we describe our studies of the N-acyl derivatives of a cyclic O-acyl hydroxamic acid, 3H-benzo[d][1,2]oxazine-1,4-dione, and, in particular, the N-tert-butoxycarbonyl derivative. This compound is not a β-lactamase inhibitor itself but undergoes spontaneous hydrolysis in aqueous solution, yielding an O-phthaloyl hydroxamic acid, which is a β-lactamase inhibitor. This compound spontaneously, but reversibly, cyclizes in solution to form phthalic anhydride, which is also a β-lactamase inhibitor. Both inhibitors react to form the same transiently stable phthaloyl–enzyme complex. Thus, we have a two-step cascade, beginning with a pro-inhibitor, in which each step leads to a different inhibitor, presumably with different enzyme specificities. The kinetics of these transformations have been elucidated in detail. The phthaloyl derivatives, where the free carboxylate is important for facile reaction with the enzyme, represent a new lead for serine β-lactamase inhibitors. Analogues can be conveniently constructed in situ by reaction of nucleophiles with phthalic anhydrides and then screened for activity. Active hits may then become new leads.

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of 5 at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl of 5 may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

Penicillin-binding proteins (PBPs) are important bacterial enzymes that carry out the final steps of bacterial cell wall assembly. Their DD-transpeptidase activity accomplishes the essential peptide cross-linking step of the cell wall. To date, all attempts to discover effective inhibitors of PBPs, apart from β-lactams, have not led to new antibiotics. Therefore, the need for new classes of efficient inhibitors of these enzymes remains. Guided by a computational fragment-based docking procedure, carried out on Escherichia coli PBP5, we have designed and synthesized a series of 4-quinolones as potential inhibitors of PBPs. We describe their binding to the PBPs of E. coli and Bacillus subtilis. Notably, these compounds bind quite tightly to the essential high molecular mass PBPs.Keywords: 4-quinolone; DD-peptidase; noncovalent inhibitor; Penicillin-binding protein

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (Ki < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.

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 bacterial dd-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A–C (LMMA, -B, and -C, respectively), is believed to catalyze dd-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 dd-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) dd-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown.

The Actinomadura R39 dd-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small d-alanyl-d-alanine terminating peptides, especially those with a side chain that mimics the amino terminus of the stem peptide precursor to the bacterial cell wall. This paper describes the synthesis of (d-α-aminopimelylamino)-d-1-ethylboronic acid, designed to be a peptidoglycan-mimetic transition state analogue inhibitor of the R39 dd-peptidase. The boronate was found to be a potent inhibitor of the peptidase with a Ki value of 32 ± 6 nM. Since it binds some 30 times more strongly than the analogous peptide substrate, the boronate may well be a transition state analogue. A crystal structure of the inhibitory complex shows the boronate covalently bound to the nucleophilic active site Ser 49. The aminopimelyl side chain is bound into the site previously identified as specific for this moiety. One boronate oxygen is held in the oxyanion hole; the other, occupying the leaving group site of acylation or the nucleophile site of deacylation, appears to be hydrogen-bonded to the hydroxyl group of Ser 298. The Ser 49 oxygen appears to be hydrogen bonded to Lys 52. If it is assumed that this structure does resemble a high-energy tetrahedral intermediate in catalysis, it seems likely that Ser 298 participates as part of a proton transfer chain initiated by Lys 52 or Lys 410 as the primary proton donor/acceptor. The structure, therefore, supports a particular class of mechanism that employs this proton transfer device.

A series of substituted aryl malonamates have been prepared. These compounds are analogues of aryl phenaceturates where the amido side chain has been replaced by a retro-amide. Like the phenaceturates, these compounds are substrates of typical class A and class C β-lactamases, particularly of the latter, and of soluble DD-peptidases. The effect of substituents α to the ester carbonyl group on turnover by these enzymes is similar to that in the phenaceturates. On the other hand, N-alkylation of the side chain amide of malonamates, but not of phenaceturates, retains the susceptibility of the compounds to hydrolysis by β-lactamases. This reactivity is not enhanced, however, by bridging the amide nitrogen and Cα atoms. A phosphonate analogue of the malonamates was found to be an irreversible inhibitor of the β-lactamases. These results, therefore, provide further evidence for the covalent access of compounds bearing retro-amide side chains to the active sites of β-lactam-recognizing enzymes.

The β-lactam-recognizing enzymes (BLRE) make up a superfamily of largely bacterial proteins that include, principally, the dd-peptidases and β-lactamases. The former enzymes catalyze the final step in bacterial cell wall biosynthesis and are inhibited by β-lactam antibiotics, while the latter enzymes catalyze the hydrolytic destruction of β-lactams and represent a major source of bacterial resistance to these antibiotics. The active site of this superfamily of enzymes includes a Ser1/Ser2(Tyr)/Lys1(His)/Lys2 tetrad in which Ser1 is a nucleophilic catalyst that becomes acylated in the formation of an acyl−enzyme intermediate. An oxyanion hole is also present. The amidase signature (AS) enzymes represent another serine amidohydrolase superfamily with no overall structural resemblance to the BLRE. The active site is characterized by a Ser1/Ser2/Lys1/NH tetrad and an oxyanion hole. We point out that there is a close spatial overlap between the two tetrads and speculate that this has arisen from a process of convergent evolution driven by a mechanistic imperative. Conversion of the backbone NH group of the AS tetrad into Lys2 of the BLRE is rationalized and leads to another mechanistic possibility that may dominate BLRE catalysis. The active site triads of other serine amidohydrolases are also briefly and comparatively discussed.

O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure−activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both d- and l-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and d-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.

A series of diaroyl phosphates was employed to assess the general reactivity of this class of molecule against classical class A and class C β-lactamases. The compounds were found, in general, to be inhibitory substrates of both classes of enzyme. In each case, they reacted rapidly with the enzyme (104 to 106 s−1 M−1) to yield transiently stable intermediates, most likely acyl-enzymes, which slowly (10−3 to 10−1 s−1) regenerated free enzyme. In certain cases, side branches from direct turnover produced EII complexes (“substrate” inhibition), more inert EI′ complexes, and, in one case, a completely inactive EI′ complex. Deacylation, but not acylation, was enhanced by electron-withdrawing substituents. Acylation rates were enhanced by hydrophobic substitution, both in the diaroyl phosphate and at the enzyme active site. The latter factor led to the general order of β-lactamase acylation rates: class D (previous results) > class C > class A. It is likely that nanomolar inhibitors of all serine β-lactamases could be achieved by rational exploitation of diacyl phosphates.

Asymmetric diaroyl phosphates (ArCOOPO2−OCOAr′, where Ar = Ph, Ar′ = 4-biphenyl, 2-benzothiophenyl and 2-benzofuranyl) have been prepared, evaluated as serine (classes A, C, and D) β-lactamase inhibitors, and compared with respect to the latter with their symmetric parents, where Ar = Ar′. The asymmetric compounds, in general, were found to react with the β-lactamases in two modes, corresponding to different orientations with respect to the active site, whereby either of the two aroyl groups may acylate the enzyme to form two different inert acyl-enzymes, E-COAr and E-COAr′ . In all cases, the asymmetric compounds, in one orientation, react more rapidly with the enzymes tested than one symmetrical parent but not both. From comparisons of activation free energy differences, it was found that the changes in free energy on changing from one aryl group to another, in either the acyl group or the leaving group, were not additive, i.e., that the effect of changing one aroyl group to another depended on the leaving group and vice versa. Thus, intramolecular cooperativity between the aroyl groups must exist, arising either from direct interaction between them or from protein-mediated interaction or from a combination of both. Such cooperativity brings fresh opportunities and challenges to the search for novel β-lactamase inhibitors.

The class C serine β-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by O-aryloxycarbonyl hydroxamates. A series of these new inhibitors has been prepared to investigate the kinetics and mechanism of the inactivation reaction. A pH−rate profile for the reaction indicated that the reactive form of the inhibitor is neutral rather than anionic. The reaction rate is enhanced by electron-withdrawing aryloxy substituents and by hydrophobic substitution on both aryloxy and hydroxamate groups. Kinetics studies show that the rates of loss of the two possible leaving groups, aryloxide and hydroxamate, are essentially the same as the rate of enzyme inactivation. Nucleophilic trapping experiments prove, however, that the aryl oxide is the first to leave. It is likely, therefore, that the rate-determining step of inactivation is the initial acylation reaction, most likely of the active site serine, yielding a hydroxamoyl-enzyme intermediate. This then partitions between hydrolysis and aminolysis by Lys 315, the latter to form an inactive, cross-linked active site. A previously described crystal structure of the inactivated enzyme shows a carbamate cross-link of Ser 64 and Lys 315. Structure−activity studies of the reported compounds suggest that they do not react at the enzyme active site in the same way as normal substrates. In particular, it appears that the initial acylation by these compounds does not involve the oxyanion hole, an unprecedented departure from known and presumed reactivity. Molecular modeling suggests that an alternative oxyanion hole may have been recruited, consisting of the side chain functional groups of Tyr 150 and Lys 315. Such an alternative mode of reaction may lead to the design of novel inhibitors.

All three classes of serine β-lactamases are inhibited at micromolar levels by 1:1 complexes of catechols with vanadate. Vanadate reacts with catechols at submillimolar concentrations in aqueous buffer at neutral pH in several steps, initially forming 1:1, 1:2, and, possibly, 1:3 complexes. Formation of these complexes is followed by the slower reduction of vanadate (VV) to vanadyl (VIV) and oxidation of the catechol. Vanadyl−catechol complexes, however, do not inhibit the β-lactamases. Rate and equilibrium constants of formation of the 1:1 and 1:2 complexes of vanadate with catechol itself and with 2,3-dihydroxynaphthalene were measured by stopped-flow spectrophotometry. Typical examples of all three classes of serine β-lactamases (the class A TEM-2, class C P99, and class D OXA-1 enzymes) were competitively inhibited by the 1:1 vanadate−catechol complexes. The inhibition was modestly enhanced by hydrophobic substituents on the catechol. The 1:1 vanadate complexes are considerably better inhibitors of the P99 β-lactamase than 1:1 complexes of catechol with boric acid and are likely to contain penta- or hexacoordinated vanadium rather than tetracooordinated. Molecular modeling showed that a pentacoordinated 1:1 vanadate−catechol complex readily fits into the class C β-lactamase active site with coordination to the nucleophilic serine hydroxyl oxygen. Such complexes may resemble the pentacoordinated transition states of phosphyl transfer, a reaction also catalyzed by β-lactamases.

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.

The membrane-bound bacterial d-alanyl-d-alanine peptidases or penicillin-binding proteins (PBPs) catalyze the final transpeptidation reaction of bacterial cell wall biosynthesis and are the targets of β-lactam antibiotics. Rather surprisingly, the substrate specificity of these enzymes is not well understood. In this paper, we present measurements of the reactivity of typical examples of these enzymes with peptidoglycan-mimetic β-lactams under in vivo conditions. The minimum inhibitory concentrations of β-lactams with Escherichia coli-specific side chains were determined against E. coli cells. Analogous measurements were made with Streptococcus pneumoniae R6. The reactivity of the relevant β-lactams with E. coli PBPs in membrane preparations was also determined. The results show that under none of the above protocols were β-lactams with peptidoglycan-mimetic side chains more reactive than generic analogues. This suggests that in vivo, as in vitro, these enzymes do not specifically recognize elements of peptidoglycan structure local to the reaction center. Substrate recognition must thus involve extended structure.

The monobactam sodium 3-benzylcarbamoyl-2-oxo-1-azetidinesulfonate, bearing a retro (vs classical β-lactam)-amide side chain, has been synthesized and the kinetics of its reaction with typical β-lactamases studied. The new compound is generally a poorer substrate than the analogous compound with a normal side chain but its formation of a transiently stable complex with a class C β-lactamase sustains the retro-amide side-chain concept.The monobactam sodium 3-benzylcarbamoyl-2-oxo-1-azetidinesulfonate, bearing a retro (vs classical β-lactam)-amide side chain, has been synthesized and the kinetics of its reaction with typical β-lactamases studied. The new compound is generally a poorer substrate than the analogous compound with a normal side chain but its formation of a transiently stable complex with a class C β-lactamase sustains the retro-amide side-chain concept.

6- and 7-Carboxy-3-phenylacetamido-3H-1-benzofuran-2-one have been synthesized as potential β-lactamase substrates and/or inhibitors. These compounds were prepared by lactonization of the corresponding, appropriately substituted phenylglycines. The latter compounds were prepared by either the Strecker or the Bücherer–Berg method. The benzofuran-2-ones were less stable in aqueous solution than the analogous acyclic phenaceturate esters but comparably stable to analogous benzopyran-2-ones. They differed from the latter compounds however in that the C-3 hydrogen of the furan-2-ones, adjacent to the lactone carbonyl group, was distinctly acidic; 7-carboxy-3-phenylacetamido-3H-1-benzofuran-2-one exists largely as an enolate at pH 7.5. The furan-2-ones were β-lactamase substrates with reactivity very similar to the analogous acyclic phenaceturates. They were not, however, dd-peptidase inhibitors and are thus unlikely to have antibiotic activity. The structural basis for these observations is discussed.Graphic

O-Aryloxycarbonyl hydroxamates and 1,3,4-oxathiazol-2-ones have been identified as covalent inhibitors of β-lactamases and proteasomes, respectively. The products of these inhibition reactions are remarkably similar, involving carbonyl cross-linking of the active sites. We have cross-checked these inhibitors, showing that the former inhibit proteasomes and the latter β-lactamases, to form the same inactive carbonyl adducts. These results are discussed in terms of similarities of the active site structures and catalytic mechanisms. It is likely that a mechanistic imperative has led to convergent evolution of these enzyme active sites, of a β-lactam-recognizing enzyme and a N-terminal protease belonging to different amidohydrolase superfamilies.Highlights► A cross-check of two inhibitors shows both inhibit a β-lactamase and a proteasome. ► Similar inhibition mechanisms and similar products. ► Active site homology but enantiotopic. ► Convergent evolution?

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with β-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 dd-peptidase structures reveal the presence of a specific binding site for the d-α-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 dd-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate–analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.

•New chromophoric substrates of mandelamide hydrolase (MAH).•Two reactive orientations of substrates at the MAH active site.•Interpretation of MAH substrate specificity in terms of MAH structure.•Inhibition of MAH by O-acyl hydroxamates.The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an “inverse” acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (Ki = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors.Download high-res image (177KB)Download full-size image

•Potent covalent inhibitors of penicillin acylase.•Two orientations of inhibitors binding to the active site.•Two distinct acyl-enzyme intermediates, one leading to hydrolysis, the other to enzyme inactivation.•Asymmetric carbonates as very versatile enzyme inhibitors.O-Aryloxycarbonyl hydroxamates have previously been shown to covalently inactivate serine/amine amidohydrolases such as class C β-lactamases and a N-terminal hydrolase, the proteasome. We report here reactions between O-aryloxycarbonyl hydroxamates and another N-terminal hydrolase, penicillin acylase. O-Aryloxycarbonyl hydroxamates, as non-symmetric carbonates, have two different leaving groups attached to the reactive central carbonyl group. We propose that these compounds can bind to the active site in either of two orientations and that either leaving group can be displaced from either orientation. In the present case we detected from kinetics experiments two distinct acyl-enzymes, one of which is subject to normal hydrolysis and the other to inactivation. Non-symmetric carbonates therefore can be very versatile enzyme inactivators.Download high-res image (90KB)Download full-size image

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of 5 at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl of 5 may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.