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
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
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DOI：
10.1074/jbc.m404603200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Sugiyama, M
中科院分区：
文献类型：
--
作者：
Noda, M;Kawahara, Y;Sugiyama, M
抗生素D-环丝氨酸（DCS）抑制细菌细胞壁生物合成所必需的丙氨酸消旋酶（ALR）和D-丙氨酰-D-丙氨酸连接酶（ALR）的催化活性。在这项研究中，我们克隆了两个基因编码ALR和ALR，指定的alrS和ddlS，分别从DCS生产链霉菌lagenulae ATCC 25233。将每个基因产物纯化至同质并表征。用携带alrS或ddlS的pET载体转化大肠杆菌，其对DCS的抗性比携带E. coli ALR或DDL编码的基因插入pET载体。虽然S.熏衣草DDL被DCS竞争性抑制，K-i值（920 μ M）明显高于E（40相似于100倍）。coli DdlA（9 μ M）或DdlB（27 μ M）。S. laaculaelae的研究表明，这种酶可能是产DCS微生物中的一种自身抗性决定因子。对S. laaculae ALR的结果表明，DCS对该酶的时间依赖性失活速率绝对慢于E. coli ALR.因此，产DCS的S.乳酸菌也是自抗性的决定因素之一。
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 muM) was obviously higher (40similar to100-fold) than those for E. coli DdlA (9 muM) or DdlB (27 muM). 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.