Lozano M J, Remsing L L, Quirós L M, Braña A F, Fernández E, Sánchez C, Méndez C, Rohr J, Salas J A
Departamento de Biología Funcional e Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain.
J Biol Chem. 2000 Feb 4;275(5):3065-74. doi: 10.1074/jbc.275.5.3065.
A DNA chromosomal region of Streptomyces argillaceus ATCC 12596, the producer organism of the antitumor polyketide drug mithramycin, was cloned. Sequence analysis of this DNA region, located between four mithramycin glycosyltransferase genes, showed the presence of two genes (mtmMI and mtmMII) whose deduced products resembled S-adenosylmethionine-dependent methyltransferases. By independent insertional inactivation of both genes nonproducing mutants were generated that accumulated different mithramycin biosynthetic intermediates. The M3DeltaMI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3DeltaM2 (mtmMII-minus mutant) accumulated 9-demethylpremithramycin A3 (9-DPMA3), premithramycin A1 (PMA1), and 7-demethylmithramycin, all of them containing the O-methyl group at C-4 and C-1', respectively, but lacking the methyl group at the aromatic position. Both genes were expressed in Streptomyces lividans TK21 under the control of the erythromycin resistance promoter (ermEp) of Saccharopolyspora erythraea. Cell-free extracts of these clones were precipitated with ammonium sulfate (90% saturation) and assayed for methylation activity using different mithramycin intermediates as substrates. Extracts of strains MJM1 (expressing the mtmMI gene) and MJM2 (expressing the mtmMII gene) catalyzed efficient transfer of tritium from [(3)H]S-adenosylmethionine into 4-DPMC and 9-DPMA3, respectively, being unable to methylate other intermediates at a detectable level. These results demonstrate that the mtmMI and mtmMII genes code for two S-adenosylmethionine-dependent methyltransferases responsible for the 4-O-methylation and 9-C-methylation steps of the biosynthetic precursors 4-DPMC and 9-DPMA3, respectively, of the antitumor drug mithramycin. A pathway is proposed for the last steps in the biosynthesis of mithramycin involving these methylation events.
产抗肿瘤聚酮类药物光神霉素的阿氏链霉菌ATCC 12596的一个DNA染色体区域被克隆。对位于四个光神霉素糖基转移酶基因之间的这个DNA区域进行序列分析，结果显示存在两个基因（mtmMI和mtmMII），其推导产物类似于依赖S-腺苷甲硫氨酸的甲基转移酶。通过对这两个基因进行独立的插入失活，产生了不产抗生素的突变体，这些突变体积聚了不同的光神霉素生物合成中间体。M3DeltaMI突变体（mtmMI缺失突变体）积累了在碳4和碳9处缺乏甲基的4-去甲基前光神霉素酮（4-DPMC）。M3DeltaM2（mtmMII缺失突变体）积累了9-去甲基前光神霉素A3（9-DPMA3）、前光神霉素A1（PMA1）和7-去甲基光神霉素，它们分别在C-4和C-1'处含有O-甲基，但在芳香位置缺乏甲基。这两个基因在红色糖多孢菌的红霉素抗性启动子（ermEp）控制下在变铅青链霉菌TK21中表达。这些克隆的无细胞提取物用硫酸铵（90%饱和度）沉淀，并用不同的光神霉素中间体作为底物测定甲基化活性。菌株MJM1（表达mtmMI基因）和MJM2（表达mtmMII基因）的提取物分别催化氚从[(3)H]S-腺苷甲硫氨酸高效转移到4-DPMC和9-DPMA3中，无法在可检测水平上使其他中间体甲基化。这些结果表明，mtmMI和mtmMII基因分别编码两种依赖S-腺苷甲硫氨酸的甲基转移酶，它们分别负责抗肿瘤药物光神霉素生物合成前体4-DPMC和9-DPMA3的4-O-甲基化和9-C-甲基化步骤。提出了一个涉及这些甲基化事件的光神霉素生物合成最后步骤的途径。
