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J. Biol. Chem., Vol. 279, Issue 44, 46143-46152, October 29, 2004

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Self-protection Mechanism in D-Cycloserine-producing Streptomyces lavendulae

GENE CLONING, CHARACTERIZATION, AND KINETICS OF ITS ALANINE RACEMASE AND D-ALANYL-D-ALANINE LIGASE, WHICH ARE TARGET ENZYMES OF D-CYCLOSERINE^*

Masafumi Noda, Yumi Kawahara, Azusa Ichikawa, Yasuyuki Matoba, Hiroaki Matsuo, Dong-Geun Lee, Takanori Kumagai, and Masanori Sugiyama

From the Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-Ku, Hiroshima 734-8551, Japan

An antibiotic, D-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and D-alanyl-D-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K_i value (920 µM) was obviously higher (40100-fold) than those for E. coli DdlA (9 µM) or DdlB (27 µM). The high K_i value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.

Received for publication, April 26, 2004 , and in revised form, August 3, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB176675 and AB176676.

^* This work was supported by the National Project on Protein Structural and Functional Analysis, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Dermatology, Shimane Medical University, 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan.

To whom correspondence should be addressed. Tel.: 81-82-257-5280; Fax: 81-82-257-5284; E-mail: sugi{at}hiroshima-u.ac.jp.

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				M. Noda, Y. Matoba, T. Kumagai, and M. Sugiyama Structural Evidence That Alanine Racemase from a D-Cycloserine-producing Microorganism Exhibits Resistance to Its Own Product J. Biol. Chem., October 29, 2004; 279(44): 46153 - 46161. [Abstract] [Full Text] [PDF]