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Volume 271, Number 47, Issue of November 22, 1996 pp. 30256-30262
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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An Alkaline D-Stereospecific Endopeptidase with -Lactamase Activity from Bacillus cereus

(Received for publication, July 22, 1996, and in revised form, August 24, 1996)

From the Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-03, Japan

We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B. The M_r of the enzyme was 37,952, and it was composed of a single polypeptide chain. The optimal pH for activity was ~10.3. The enzyme was strictly D-stereospecific toward oligopeptides composed of Dphenylalanine such as (D-Phe)₃ and (D-Phe)₄. The enzyme also acted to a lesser extent on (D-Phe)₆, Boc-(D-Phe)₄ (where Boc is tert-butoxycarbonyl), Boc-(D-Phe)₄ methyl ester, Boc-(D-Phe)₃ methyl ester, Boc-(D-Phe)₂, (D-Phe)₂, and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)_n (n = 2-5), (D-Val)₃, and (D-Leu)₂. The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)₂-D-Tyr and D-Tyr-(D-Phe)₂) and eight stereoisomers of (Phe)₃. The enzyme had -lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable. The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene. The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C -lactamases. Thus, the enzyme was categorized as a new "penicillin-recognizing enzyme."

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