Structure of the wall peptidoglycan of streptomyces R39 and the specificity profile of its exocellular DD-carboxypeptidase-transpeptidase for peptide acceptors

in Biochemistry (1973), 12(7), 1243-1251

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the ... [more ▼]

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl2 buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10−6, 1.5×10−6 and 0.63×10−6s−1 (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [14C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [14C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10−5s−1 and 0.8×10−4s−1 respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site. [less ▲]

Transpeptidase activity of Streptomyces D-alanyl-D carboxypeptidases

in Proceedings of the National Academy of Sciences of the United States of America (1972), 69(3), 662-666

In the presence of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala as donor, and either D-[(14)C]alanine, [(14)C]-glycine, or meso-[(3)H]diaminopimelic acid as acceptor, the DD carboxypeptidases from ... [more ▼]

In the presence of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-Ala as donor, and either D-[(14)C]alanine, [(14)C]-glycine, or meso-[(3)H]diaminopimelic acid as acceptor, the DD carboxypeptidases from Streptomyces R61 and R39 catalyze a transpeptidation reaction with the release of terminal D-alanine from the donor and the formation of either N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-[(14)C]Ala, N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-[(14)C] Gly, or N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala-D-meso- [(3)H]diaminopimelic acid. The reaction appears to be a true transpeptidation, and is not simply a "reversal of hydrolysis". Transpeptidation is inhibited by pencillin at concentrations that inhibit hydrolysis (carboxypeptidase action) of the donor peptide. There are differences in the specificity profiles of the Streptomyces enzymes for acceptor molecules:only the R61 enzyme used [(14)C]Gly-Gly as acceptor; transfer of N(alpha),N(epsilon)-diacetyl-L-Lys-D-Ala to this acceptor resulted in the formation of N(alpha),N(epsilon)-diacetyl-Lys-D-Ala-[(14)C] Gly-Gly, with the synthesis of a (D-Ala-Gly) peptide bond in an endoposition. [less ▲]