Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699

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• 作者：Xu ; Jun ; Mahmud ; Taifo ; Floss ; Heinz G.
• 关键词：AdoMet ; 27-O-demethylrifamycin SV methyltransferase ; 27-O-demethylrifamycin SV ; 27-O-demethyl-25-O-desacetylrifamycin SV ; Amycolatopsis mediterranei ; rif orf14
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2003
• 期刊代码：116_00039861
• 类别：ch
• 出版时间：March 15, 2003
• 卷：411
• 期：2
• 页码：277-288
• 文件大小：351 K

摘要

The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca²⁺ or Mg²⁺ inhibits the enzyme activity slightly, Zn²⁺, Ni²⁺, and Co²⁺ are strongly inhibitory. The K_m values for DMRSV and S-adenosyl-l-methionine (AdoMet) are 18.0 and 19.3μM, respectively, and the K_cat is 87s⁻¹. The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.