With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes with a higher efficiency than the D isomer.

In the analysis of the interactions between beta-lactam antibiotics and their target enzymes, it is often difficult to estimate the kinetic properties of the molecules which react rapidly with their targets and in consequence behave as the most efficient antibiotics. The combined utilization of fluorescein-labelled penicillins and of a new competition method has allowed an accurate determination of the high second-order rate constants characterizing the acylation of Bacillus licheniformis penicillin-binding protein 1 (PBP1) by penicillins and cephalosporins. Strategies were devised for measuring high acylation rates while avoiding titration effects. The method was also suitable for measuring the PBP kinetic parameters in intact cells. These results also confirmed that PBP1 is probably the main target of most beta-lactam antibiotics. Cephalexin, however, reacted faster with PBP3.

Five class A beta-lactamases produced by various mesophilic bacterial species have been compared. Although closely related in primary and overall structures, these enzymes exhibit very different stabilities. In order to investigate the factors responsible for these differences, several features deduced from the amino acid composition and three-dimensional structures were studied for the five proteins. This analysis revealed that higher stability appeared to correlate with increased numbers of intramolecular hydrogen bonds and of salt bridges. By contrast, the global hydrophobicity of the protein seemed to play a relatively minor role. A strongly unfavourable balance between charged residues and the presence of a cis-peptide bond preceding a non-proline residue might also contribute to the particularly low stability of two of the enzymes.

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3.

By using site-directed mutagenesis, the conserved Lys-67 residue situated three positions after the active-site Ser of a class C beta-lactamase was replaced by Arg or Gln. The Lys-67-Gln protein was nearly inactive. Although severely impaired, the Lys-67-Arg mutant exhibited an appreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the kcat. values were even increased. The properties of the Lys-67-Arg mutant were studied by both steady-state and transient-state kinetic methods with a variety of compounds representing distinct classes of available substrates. With beta-lactam substrates, the kcat./Km values reflecting the efficiency of the acylation step (k+2/K) were decreased 25-100-fold. When the individual values could be measured, k+2 was not significantly altered, but K was found to be strongly increased, a result most likely explained by a corresponding increase in the k+1/k-1 ratio. These results, combined with the much stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of the Lys-67 side chain.

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments.

The synthesis and properties of six fluorescein-labelled penicillins are reported. The two isomers of fluoresceyl-glycyl-6-amino-penicillanic acid are probably the best compounds to use for detection of all the penicillin-binding proteins (PBPs) present in a bacterial membrane preparation. However, the derivatives of ampicillin were much more efficient against Enterobacter aerogenes PBP3. The two isomers obtained when a commercial mixture of the two isomers of carboxyfluorescein was used most often exhibited similar properties, but the Streptomyces R61 extracellular DD-peptidase was only efficiently acylated by the 5′-carboxyfluorescein derivative of glycyl-6-aminopenicillanic acid.

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. ‘Burst’ kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two ‘bursts’, and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.

The interactions between three class A beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring (cefoxitin, moxalactam and temocillin) have been studied. When compared with the situation prevailing with good substrates, both acylation and deacylation steps appeared to be severely impaired. Molecular modelling studies of the structures of the Henri-Michaelis complexes and of the acyl-enzymes indicate a major displacement of the crystallographically observed water molecule which connects the glutamate-166 and serine-70 side chains and underline the role of this water molecule in both reaction steps.

The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.

A beta-lactamase produced by Pseudomonas stutzeri was purified to protein homogeneity, and its physicochemical and catalytic properties were determined. Its profile was unusual since, in addition to penicillins, the enzyme hydrolysed second- and third-generation ‘beta-lactamase-stable’ cephalosporins and monobactams with similar efficiencies. On the basis of the characteristics of the interaction with beta-iodopenicillanic acid, the enzyme could be classified as a class-A beta-lactamase. However, when compared with most class-A beta-lactamases, it exhibited significantly lower kcat./Km values for the compounds usually considered to be the best substrates of these enzymes.

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains.

A cysteine residue has been substituted for the active-site serine of the class-C beta-lactamase produced by Enterobacter cloacae 908R by site-directed mutagenesis. The modified protein exhibited drastically reduced kcat./Km values on all tested substrates. However, this decrease was due to increased Km values with some substrates and to decreased kcat. values with others. These apparently contradictory results could be explained by a selective influence of the mutation on the first-order rate constant characteristic of the acylation step, a hypothesis which was confirmed by the absence of detectable acylenzyme accumulation with all the tested substrates, with the sole exception of cefoxitin.

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding was also drastically decreased, except for the reactions with nitrocefin and methicillin. The consequences of the first mutation were more surprising, since a large proportion of the thiolesterase activity was retained, together with the penicillin-binding capacity. Conversely, the peptidase properties was severely affected. In both cases, a drastic decrease in the transferase activity was observed. The results are compared with those obtained by mutation of the corresponding residue in the class A beta-lactamase of Streptomyces albus G.

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific ‘carbapenemase’. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column.

A new method for the identification and quantification of penicillin-binding proteins is described which uses fluorescein-coupled penicillins. It allows the rapid detection of 0.2 pmol with the naked eye and 2 fmol with the help of an A.L.F. automatic DNA sequencer. Direct labelling can also be performed on whole bacterial cells.

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia coli PBP4 [Mottl, Terpstra & Keck (1991) FEMS Microbiol. Lett. 78, 213-220]. Hydrophobic-cluster analysis of the two proteins shows that, providing that a large 174-amino-acid stretch is excluded from the analysis, the bulk of the two polypeptide chains possesses homologues of the active-site motifs and secondary structures found in the class A beta-lactamase of Streptomyces albus G of known three-dimensional structure. The 174-amino-acid insert occurs at equivalent places in the two PBPs, between helices alpha 2 and alpha 3, away from the active site. Such an insert is unique among the penicilloyl serine transferases. It is proposed that the Actinomadura R39 PBP and E. coli PBP4 form a special class, class C, of low-Mr PBPs/DD-peptidases. A vector has been constructed and introduced by electrotransformation in the original Actinomadura R39 strain, allowing high-level expression and secretion of the DD-peptidase/PBP (250 mg.l-1). The gene encoding the desired protein is processed differently in Actinomadura R39 and Streptomyces lividans. Incorrect processing in Streptomyces lividans leads to a secreted protein which is inert in terms of DD-peptidase activity and penicillin-binding capacity.

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

Among the active-site-serine penicillin-recognizing proteins, the Streptomyces R61 extracellular DD-peptidase is the only one to have a His-Thr-Gly sequence [instead of Lys-Thr(Ser)-Gly] in ‘box’ VII. The His residue was replaced by Gln or Lys. Both mutations induced a marked decrease in the rates of both tripeptide substrate hydrolysis and acylation by benzylpenicillin and cephalosporin C. The rate of hydrolysis of the thioester hippuryl thioglycollate was less affected. The most striking result was the disproportionate loss of transpeptidation properties by both mutants, indicating an important role of His-298 in this reaction. We believe that this result represents the first modification of a DD-peptidase leading to a specific decrease of the transpeptidation-to-hydrolysis ratio.

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

Thioester substrates can be used to study the hydrolysis and transfer reactions catalysed by beta-lactamases and DD-peptidases. With the latter enzymes, accumulation of the acyl-enzyme can be detected directly. The efficiency of various amines as acceptor substrates was in excellent agreement with previous results obtained with peptide substrates of the DD-peptidases. The results indicated the presence of a specific binding site for the acceptor substrates. Although most of the results agreed well with a simple partition model, more elaborate hypotheses will be needed to account for all the data presented.

The 29,000-Mr Actinomadura R39 beta-lactamase exhibited a remarkably low electrophoretic mobility on SDS/PAGE, yielding an Mr value almost twice that computed from the corresponding gene sequence. We showed that chemical modification of the carboxylic groups of glutamic acid and aspartic acid residues restored a normal electrophoretic mobility and that the anomalous behaviour of that protein on SDS/PAGE was due to its very large negative charge at neutral pH. We also compared the behaviour of the same enzyme on gel filtration in the presence of SDS with those of other class A beta-lactamases (Mr approx. 30,000). These experiments suggested that the very low electrophoretic mobility of the Actinomadura R39 beta-lactamase upon SDS/PAGE was more probably due to a low degree of SDS binding rather than to an unusual shape of the SDS-protein complex.

Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, Christensen & Waley (1990) Biochem J. 272, 613-619].

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated.

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A.

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of Actinomadura R39 beta-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the ‘heterogeneous’ preparations.

A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined. On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates. The enzyme also hydrolysed ureidopenicillins and some so-called ‘beta-lactamase-stable’ cephalosporins.

Ser130, Asp131 and Asn132 (‘SDN’) are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

Various ester and thioester derivatives of hippuric acid have been prepared which were substrates of both beta-lactamases and DD-peptidases. The thioesters were more rapidly hydrolysed by nearly all the enzymes. Surprisingly, the enzymes acted rather efficiently on substrates which did not contain any chiral centre.

The catalytic properties of four class A beta-lactamases were studied with 24 different substrates. They exhibit a wide range of variation. Similarly, the amino acid sequences are also quite different. However, no relationships were found between the sequence similarities and the substrate profiles. Lags and bursts were observed with various compounds containing a large sterically hindered side chain. As a group, the enzymes could be distinguished from the class C beta-lactamases on the basis of the kappa cat. values for several substrates, particularly oxacillin, cloxacillin and carbenicillin. Surprisingly, that distinction was impossible with the kappa cat./Km values, which represent the rates of acylation of the active-site serine residue by the beta-lactam. For several cephalosporin substrates (e.g. cefuroxime and cefotaxime) class A enzymes consistently exhibited higher kappa cat. values than class C enzymes, thus belying the usual distinction between ‘penicillinases’ and ‘cephalosporinases’. The problem of the repartition of class A beta-lactamases into sub-classes is discussed.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.

Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates of six class C beta-lactamases. Nitrocefin, cephaloridine, cefazolin, cephalothin and cephalexin were good substrates, with kcat. values ranging from 27 to 5000 s-1. Cefuroxime, cefotaxime and cefoxitin exhibited low kcat. values (0.010-1.7 s-1) and low Km values, which suggested a rate-limiting deacylation. Imipenem and aztreonam were even poorer substrates (kcat. 2 x 10(-4)-3 x 10(-2) s-1) and, in the presence of a reporter substrate, behaved as transient inactivators. With moxalactam, biphasic kinetics were observed, indicating a possible rearrangement of the acyl-enzyme.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.

Two chromosome-encoded beta-lactamases have been purified from Citrobacter diversus ULA-27. They exhibited slightly different isoelectric points (6.8 and 6.2) and very similar Mr values (congruent to 29,000). Their specificity spectrum was rather wide, since they hydrolysed some cephalosporins with kcat: values similar to those observed with the best penicillin substrates. Cloxacillin, methicillin and imipenem were hydrolysed very slowly. Hydrolysis of azthreonam could not be detected.

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae.

The sequences of three Enterobacter cloacae ampC beta-lactamase genes have been determined. The deduced amino acid sequences are very similar: out of a total of 361 residues, only eight positions were found to be variable, and several mutations yielded residues with very similar properties. The kinetic properties of two of the enzymes were not significantly different. The three enzymes also exhibited a high degree of homology (greater than 70%) with the ampC beta-lactamases of Escherichia coli K12 and Citrobacter freundii, confirming the homogeneity of class-C beta-lactamases.

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.

The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods. The enzyme is a typical two-domain protein. One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet. The active-site serine residue (Ser-48) is within a cleft located between the two domains.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.