Molecular analysis of beta-lactamases from four species of Streptomyces: comparison of amino acid sequences with those of other beta-lactamases

Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.     

Evidence for an oxyanion hole in serine beta-lactamases and DD-peptidases.

A thionocephalosporin is shown to be a much poorer substrate of representative serine beta-lactamases of class A (RTEM-2) and class C (Enterobacter cloacae P99) and a much poorer inhibitor of the Streptomyces R61 DD-peptidase than is the analogous oxo beta-lactam. These results provide kinetic evidence for the existence of a catalytic oxyanion hole in these enzymes.     

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue.     

The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family.

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.     

Active sites of beta-lactamases. The chromosomal beta-lactamases of Pseudomonas aeruginosa and Escherichia coli.

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.     

Inhibition of class C beta-lactamases by (1''R,6R)-6-(1''-hydroxy)benzylpenicillanic acid SS-dioxide.

beta-Lactamases, enzymes that catalyse the hydrolysis of the beta-lactam ring in beta-lactam antibiotics, are divided into three classes, A, B and C, on the basis of the structures so far determined. There are relatively few effective inhibitors of class C beta-lactamases. A beta-lactam sulphone with a hydroxybenzyl side chain, namely (1'R,6R)-6-(1'-hydroxy)benzylpenicillanic acid SS-dioxide (I), has now been studied. The sulphone is a good mechanism-based inhibitor of class C beta-lactamases. At pH8, the inhibition of a Pseudomonas beta-lactamase is irreversible, and proceeds at a rate that is about one-tenth the rate of concurrent hydrolysis. The labelled enzyme has enhanced u.v. absorption and is probably an enamine. At a lower pH, however, inhibition is transitory.     

Primary structure of the Streptomyces R61 extracellular DD-peptidase. 1. Cloning into Streptomyces lividans and nucleotide sequence of the gene

An 11,450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptomyces R61 was cloned in Streptomyces lividans using the high-copy-number plasmid pIJ702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DD-peptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine beta-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coli ampC beta-lactamase (class C). Since the Streptomyces R61 DD-peptidase and beta-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the beta-lactamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the beta-lactamases of classes A and C are related in an evolutionary sense.     

The diversity of the catalytic properties of class A beta-lactamases.

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.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.     

Insights into class D beta-lactamases are revealed by the crystal structure of the OXA10 enzyme from Pseudomonas aeruginosa

BACKGROUND: beta-lactam antibiotic therapies are commonly challenged by the hydrolytic activities of beta-lactamases in bacteria. These enzymes have been grouped into four classes: A, B, C, and D. Class B beta-lactamases are zinc dependent, and enzymes of classes A, C, and D are transiently acylated on a serine residue in the course of the turnover chemistry. While class A and C beta-lactamases have been extensively characterized by biochemical and structural methods, class D enzymes remain the least studied despite their increasing importance in the clinic. RESULTS: The crystal structure of the OXA10 class D beta-lactamase has been solved to 1.66 A resolution from a gold derivative and MAD phasing. This structure reveals that beta-lactamases from classes D and A, despite very poor sequence similarity, share a similar overall fold. An additional beta strand in OXA10 mediates the association into dimers characterized by analytical ultracentrifugation. Major differences are found when comparing the molecular details of the active site of this class D enzyme to the corresponding regions in class A and C beta-lactamases. In the native structure of the OXA10 enzyme solved to 1.8 A, Lys-70 is carbamylated. CONCLUSIONS: Several features were revealed by this study: the dimeric structure of the OXA10 beta-lactamase, an extension of the substrate binding site which suggests that class D enzymes may bind other substrates beside beta-lactams, and carbamylation of the active site Lys-70 residue. The CO2-dependent activity of the OXA10 enzyme and the kinetic properties of the natural OXA17 mutant protein suggest possible relationships between carbamylation, inhibition of the enzyme by anions, and biphasic behavior of the enzyme.     

Cephalosporin-derived inhibitors of beta-lactamase. Part 4: The C3 substituent

New C3-substituted beta-lactamase inhibitors were prepared and evaluated against representative class A and class C enzymes. It was possible to improve simultaneous inhibitory activity of both classes of serine hydrolase. Other inhibitors showed high selectivity for either the class C cephalosporinases or the class A penicillinases. This represents the first time that cephalosporin-derived inhibitors have demonstrated selectivity for the class A beta-lactamases.     

Why clinically used tazobactam and sulbactam are poor inhibitors of OXA-10 beta-lactamase: Raman crystallographic evidence

The clinically used inhibitors tazobactam and sulbactam are effective in the inhibition of activity of class A beta-lactamases, but not for class D beta-lactamases. The two inhibitors exhibit a complex multistep profile for their chemistry of inhibition with class A beta-lactamases. To compare the inhibition profiles for class A and D enzymes, the reactions were investigated within OXA-10 beta-lactamase (a class D enzyme) crystals using a Raman microscope. The favored reaction pathway appears to be distinctly different from that for class A beta-lactamases. In contrast to the case of class A enzymes that favor the formation of a key enamine species, the OXA-10 enzyme forms an alpha,beta-unsaturated acrylate (acid or ester). Quantum mechanical calculations support the likely product as the adduct of Ser115 to the acrylate. Few enamine-like species are formed by sulbactam or tazobactam with this enzyme. Taken together, our results show that the facile conversion of the initial imine, formed upon acylation of the active site Ser67, to the cis- and/or trans-enamine is disfavored. Instead, there is a significant population of the imine that could either experience cross-linking to a second nucleophile (e.g., Ser115) or give rise to the alpha,beta-unsaturated product and permanent inhibition. Alternatively, the imine can undergo hydrolysis to regenerate the catalytically active OXA-10 enzyme. This last process is the dominant one for class D beta-lactamases since the enzyme is not effectively inhibited. In contrast to sulbactam and tazobactam, the reactions between oxacillin or 6alpha-hydroxyisopropylpenicillinate (both substrates) and OXA-10 beta-lactamase appear much less complex. These compounds lead to a single acyl-enzyme species, the presence of which was confirmed by Raman and MALDI-TOF experiments.     

Phenylpropynal, a specific, irreversible, non-beta-lactam inhibitor of beta-lactamases

Phenylpropynal is a specific, irreversible, non-beta-lactam inhibitor of typical beta-lactamases. In the presence of millimolar concentrations of phenylpropynal, the beta-lactamase I of Bacillus cereus and the beta-lactamases of Staphylococcus aureus and Escherichia coli become completely inactivated; the beta-lactamase II of B. cereus is not affected. The E. coli beta-lactamase is considerably more sensitive to the reagent than the gram-positive enzymes. A variety of structural analogs of phenylpropynal are much less effective inhibitors. Bovine alpha-chymotrypsin, bovine carboxypeptidase A, and the D,D-carboxypeptidase/transpeptidase of Streptomyces R-61 were not inactivated by phenylpropynal. The inactivation of the E. coli beta-lactamase can be significantly retarded when the good substrate benzylpenicillin is also present. The development of a characteristic chromophore (lambda max 318 nm) during beta-lactamase inactivation suggests that covalent modification of the enxymes is involved; arginine and/or lysine modification is indicated.     

Inverse acyl phosph(on)ates: substrates or inhibitors of beta-lactam-recognizing enzymes?

Acyl phosph(on)ates represent a new class of inhibitors of beta-lactam-recognizing enzymes. Previously described members of this class were aroyl phosph(on)ates. These compounds have been shown to acylate and/or phosphylate the active site serine residue, leading to either transient or essentially irreversible inhibition [Li, N., and Pratt, R. F. (1998) J. Am. Chem. Soc.120, 4264-4268]. The present paper describes the synthesis and evaluation as inhibitors of an inverse pair of acyl phosph(on)ates that incorporate the amido side chain that represents a major substrate specificity determinant of these enzymes. Thus, N-(phenylacetyl)glycyl phenyl phosphate and benzoyl N-(benzyloxycarbonyl)aminomethyl phosphonate were prepared. The former of these compounds was found to be a substrate of typical class A and C beta-lactamases and of the DD-peptidase of Streptomyces R61; it thus acylates the active site serine. In contrast, the latter compound was an irreversible inhibitor of the above enzymes, probably by phosphonylation of the active site serine. With each of these enzymes therefore, the amido side chain rather than the acyl group dictates the orientation of the bound phosph(on)ate and thus the mode of reaction.     

6-beta-Iodopenicillanate as a probe for the classification of beta-lactamases.

An inactivator of serine beta-lactamases, 6 beta-iodopenicillanate, can be utilized as a probe in the classification of beta-lactamases. It is a substrate for class-B Zn2+-containing beta-lactamase II. Although it inactivates enzymes from both classes A and C, it is much more efficient for the former group, with which it sometimes interacts following a branched pathway. On the basis of these observations, predictions are made concerning the class to which several enzymes belong.     

Cefotaximases (CTX-M-ases), an expanding family of extended-spectrum beta-lactamases

Among the extended-spectrum beta-lactamases, the cefotaximases (CTX-M-ases) constitute a rapidly growing cluster of enzymes that have disseminated geographically. The CTX-M-ases, which hydrolyze cefotaxime efficiently, are mostly encoded by transferable plasmids, and the enzymes have been found predominantly in Enterobacteriaceae, most prevalently in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis. Isolates of Vibrio cholerae, Acinetobacter baumannii, and Aeromonas hydrophila encoding CTX-M-ases have also been reported. The CTX-M-ases belong to the molecular class A beta-lactamases, and the enzymes are functionally characterized as extended-spectrum beta-lactamases. This group of beta-lactamases confers resistance to penicillins, extended-spectrum cephalosporins, and monobactams, and the enzymes are inhibited by clavulanate, sulbactam, and tazobactam. Typically, the CTX-M-ases hydrolyze cefotaxime more efficiently than ceftazidime, which is reflected in substantially higher MICs to cefotaxime than to ceftazidime. Phylogenetically, the CTX-M-ases are divided into four subfamilies that seem to have descended from chromosomal beta-lactamases of Kluyvera spp. Insertion sequences, especially ISEcp1, have been found adjacent to genes encoding enzymes of all four subfamilies. The class I integron-associated orf513 also seems to be involved in the mobilization of blaCTX-M genes. This review discusses the phylogeny and the hydrolytic properties of the CTX-M-ases, as well as their geographic occurrence and mode of spread.     

Studies on structure-based sequence alignment and phylogenies of beta-lactamases

The β-lactamases enzymes cleave the amide bond in β-lactam ring, rendering β-lactam antibiotics harmless to bacteria. In this communication we have studied structure-function relationship and phylogenies of class A, B and D beta-lactamases using structure-based sequence alignment and phylip programs respectively. The data of structure-based sequence alignment suggests that in different isolates of TEM-1, mutations did not occur at or near sequence motifs. Since deletions are reported to be lethal to structure and function of enzyme. Therefore, in these variants antibiotic hydrolysis profile and specificity will be affected. The alignment data of class A enzyme SHV-1, CTX-M-15, class D enzyme, OXA-10, and class B enzyme VIM-2 and SIM-1 show sequence motifs along with other part of polypeptide are essentially conserved. These results imply that conformations of betalactamases are close to native state and possess normal hydrolytic activities towards beta-lactam antibiotics. However, class B enzyme such as IMP-1 and NDM-1 are less conserved than other class A and D studied here because mutation and deletions occurred at critically important region such as active site. Therefore, the structure of these beta-lactamases will be altered and antibiotic hydrolysis profile will be affected. Phylogenetic studies suggest that class A and D beta-lactamases including TOHO-1 and OXA-10 respectively evolved by horizontal gene transfer (HGT) whereas other member of class A such as TEM-1 evolved by gene duplication mechanism. Taken together, these studies justify structure-function relationship of beta-lactamases and phylogenetic studies suggest these enzymes evolved by different mechanisms.     

Interaction of clavulanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways. Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat. = 18s-1 and 52s-1 respectively). If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed. At lower ratios, inactivation is only partial. Irreversible inactivation occurs after 400 and 20000 turnovers for the A. R39 and S. albus G enzymes respectively. With the A. R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S. albus G beta-lactamase. Kinetic models are presented and studied for the interaction between clavulanate and both enzymes. A tentative general reaction scheme is also discussed.     

The inhibition of class C beta-lactamases by boronic acids.

Aromatic boronic acids are reversible inhibitors of the recently classified class C beta-lactamases. The boronic acids studied include ortho-, meta- and para-methyl-, -hydroxymethyl- and -formyl-phenylboronic acid. The beta-lactamases were chromosomally-encoded enzymes, one from Pseudomonas aeruginosa, and the other specified by the ampC gene of Escherichia coli. The inhibition may be correlated with our finding that these beta-lactamases are serine enzymes, i.e. their function entails the hydroxy group of a serine residue acting as a nucleophile.