Kudo Fumitaka, Kawabe Kenichi, Kuriki Hisako, Eguchi Tadashi, Kakinuma Katsumi
Contribution from the Department of Chemistry and Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan.
J Am Chem Soc. 2005 Feb 16;127(6):1711-8. doi: 10.1021/ja044921b.
Aminoglycoside antibiotics are composed of aminosugars and a unique aminocyclitol aglycon including 2-deoxystreptamine (DOS), streptidine, actinamine, etc., and nucleotidylyltransferases, sugar modifying enzymes, and glycosyltransferases appear to be essential for their biosynthesis. However, the genes encoding those enzymes were unable to be identified by a standard homology search in the butirosin biosynthetic btr gene cluster, except that the btrM gene appeared to be a glycosyltransfease. Disruption studies of the btrD gene indicated that BtrD was involved in the supply of a glycosyl donor immediately prior to the glycosylation of DOS giving paromamine. As anticipated, BtrD expressed in Escherichia coli was able to catalyze UDP-D-glucosamine formation from D-glucosamine-1-phosphate and UTP. Both dTTP and UTP were good NTP substrates, and D-glucose-1-phosphate and D-glucosamine-1-phosphate were good sugar phosphates for the enzyme reaction. This finding is the first to identify an enzyme which activates a sugar donor in the DOS-containing antibiotics. Interestingly, BtrD homologues have been reported as functionally unknown open reading frames (ORFs) in the biosynthetic gene clusters for several antibiotics including teicoplanin, balhimycin, chloroeremomycin, and mitomycin C. It appears therefore that gene clusters for antibiotic biosynthesis provide their own nucleotidylyltransferases, and the BtrD homologues are among the secondary metabolism specific enzymes.
氨基糖苷类抗生素由氨基糖和独特的氨基环醇苷元组成，包括2-脱氧链霉胺（DOS）、链霉胍、放线胺等，核苷酸转移酶、糖修饰酶和糖基转移酶似乎对其生物合成至关重要。然而，除了btrM基因似乎是一种糖基转移酶外，在丁胺卡那霉素生物合成的btr基因簇中，通过标准同源性搜索无法鉴定编码这些酶的基因。对btrD基因的破坏研究表明，BtrD在DOS糖基化生成巴龙胺之前直接参与糖基供体的供应。正如预期的那样，在大肠杆菌中表达的BtrD能够催化由D-葡萄糖胺-1-磷酸和UTP形成UDP-D-葡萄糖胺。dTTP和UTP都是良好的NTP底物，D-葡萄糖-1-磷酸和D-葡萄糖胺-1-磷酸是该酶反应的良好糖磷酸酯。这一发现首次鉴定出一种在含DOS抗生素中激活糖供体的酶。有趣的是，在替考拉宁、巴利霉素、氯埃雷霉素和丝裂霉素C等几种抗生素的生物合成基因簇中，BtrD同源物已被报道为功能未知的开放阅读框（ORF）。因此，抗生素生物合成的基因簇似乎提供了它们自己的核苷酸转移酶，而BtrD同源物属于次级代谢特异性酶。
