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The mechanism of action of DD-peptidases: the role of tyrosine-159 in the Streptomyces R61 DD-peptidase.

ABSTRACT: 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.

SUBMITTER: Wilkin JM

PROVIDER: S-EPMC1132558 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Mechanism of action of DD-peptidases: role of asparagine-161 in the Streptomyces R61 DD-peptidase.

Project description: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.

| S-EPMC1134339 | biostudies-other

The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.

Project description: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.

| S-EPMC1137106 | biostudies-other

Point mutations of two arginine residues in the Streptomyces R61 DD-peptidase.

Project description: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.

| S-EPMC1131003 | biostudies-other

Importance of the His-298 residue in the catalytic mechanism of the Streptomyces R61 extracellular DD-peptidase.

Project description: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.

| S-EPMC1130807 | biostudies-other

Importance of the two tryptophan residues in the Streptomyces R61 exocellular DD-peptidase.

Project description: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.

| S-EPMC1130787 | biostudies-other

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

Project description: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.

| S-EPMC1148858 | biostudies-other

Inhibition of DD-peptidases by a specific trifluoroketone: crystal structure of a complex with the Actinomadura R39 DD-peptidase.

Project description: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.

| S-EPMC3731424 | biostudies-literature

Effects of thiol reagents on Streptomyces K15 DD-peptidase-catalysed reactions.

Project description:The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5'-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB decreases the efficacy of the initial binding of the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate to the enzyme (K), the rate of enzyme acylation by the donor (K+2) and the rate of enzyme deacylation (k+3). However, the value of the k+2/k+3 ratio, and therefore the percentage of total enzyme which, at saturating concentrations of the donor, is present as acyl-enzyme at the steady state of the reaction, are not modified. The enzyme's binding sites for pCMB and benzylpenicillin are not mutually exclusive. But, when compared with the native enzyme, the pCMB-derivatized enzyme undergoes acylation by benzylpenicillin with a decreased second-order-rate constant (k+2/K) value and gives rise to a penicilloyl adduct of increased stability. Since the acyl-enzyme mechanism is not annihilated by pCMB derivatization, it is proposed that basically, and like all the other DD-peptidases/penicillin-binding proteins so far characterized, the Streptomyces K15 DD-peptidase is an active-site-serine enzyme.

| S-EPMC1147644 | biostudies-other

Interactions of cephalosporins with the Streptomyces R61 DD-transpeptidase/carboxypeptidase. Influence of the 3'-substituent.

Project description:The influence and fate of the 3'-substituent of a series of cephalosporins on their interaction with the DD-transpeptidase/carboxypeptidase of Streptomyces R61 were investigated. Good 3'-leaving groups are eliminated from the acyl-enzyme before deacylation, and hence cephalosporins with such substituents generate a common covalent intermediate. The rate of this elimination reaction in the two cases studied in detail was much lower than from the corresponding cephalosporoates in free solution. Cephalosporins without good 3'-leaving groups yield acyl-enzymes that appear to hydrolyse, leading to enzyme re-activation, faster than those from cephalosporins with leaving groups.

| S-EPMC1147132 | biostudies-other

Molecular weight and amino acid composition of the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61.

Project description:A procedure allowing the purification of milligram amounts of the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 to protein homogeneity (95% purity) is described. The isolated protein has a molecular weight of about 38000 and consists of one polypeptide chain. Its amino acid composition is presented.

| S-EPMC1165848 | biostudies-other

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