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The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.

ABSTRACT: The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of the conserved tetrad Ser-Xaa-Xaa-Lys occurs at position 35. There is no indication for the presence of a signal peptide or an N-terminal hydrophobic sequence, suggesting that the Streptomyces K15 enzyme is probably anchored to the membrane by a C-terminal peptide segment.

SUBMITTER: Leyh-Bouille M

PROVIDER: S-EPMC1138712 | biostudies-other | 1989 Jun

REPOSITORIES: biostudies-other

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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.

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Amino acid sequence of the penicillin-binding protein/DD-peptidase of Streptomyces K15. Predicted secondary structures of the low Mr penicillin-binding proteins of class A.

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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.

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Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

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Streptomyces K15 DD-peptidase-catalysed reactions with ester and amide carbonyl donors.

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Secretion by overexpression and purification of the water-soluble Streptomyces K15 DD-transpeptidase/penicillin-binding protein.

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| S-EPMC103608 | biostudies-literature

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