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

ABSTRACT: 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.

SUBMITTER: Bourguignon-Bellefroid C

PROVIDER: S-EPMC1131003 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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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.

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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.

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

Project description: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.

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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.

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

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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.

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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.

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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.

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Nucleophilic re-activation of the PC1 beta-lactamase of Staphylococcus aureus and of the DD-peptidase of Streptomyces R61 after their inactivation by cephalosporins and cephamycins.

Project description:It has been shown previously [Faraci & Pratt (1985) Biochemistry 24, 903-910; (1986) Biochemistry 25, 2934-2941; (1986) Biochem. J. 238, 309-312] that certain beta-lactam-processing enzymes form inert acyl-enzymes with cephems that possess good leaving groups at the C-3' position. These inert species arise by elimination of the leaving group from the initially formed and more rapidly hydrolysing acyl-enzyme, which has the 'normal' cephalosporoate structure. The present paper shows that a strong nucleophile, thiophenoxide, can catalyse the re-activation of three examples of these inert acyl-enzymes, generated on reaction of cephalothin and cefoxitin with the PC1 beta-lactamase of Staphylococcus aureus and of cephalothin with D-alanyl-D-alanine transpeptidase/carboxypeptidase of Streptomyces R61. In view of the reversibility of the elimination reaction, demonstrated in model systems [Pratt & Faraci (1986) J. Am. Chem. Soc. 108, 5328-5333], this catalysis is proposed to arise through nucleophilic addition to the exo-methylene carbon atom of the inert acyl-enzyme to regenerate a more rapidly hydrolysing normal cephalosporoate. Strong support for this scenario was obtained through comparison of the kinetics of the catalysed re-activation reaction with those of turnover of the relevant 3'-thiophenoxycephems, thiophenoxycephalothin and thiophenoxycefoxitin. The enzymes appear to stabilize the products of the elimination reaction with respect to the normal cephalosporoate, but more strongly to destabilize the transition states. The effects of other nucleophiles, including cysteine, glycine amide and imidazole, on the above enzymes and on other beta-lactamases can be understood in terms of the model reaction kinetics and thermodynamics.

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