State Key Lab of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Rd., Shanghai 200040, China.
Bioorg Med Chem Lett. 2013 Mar 1;23(5):1438-41. doi: 10.1016/j.bmcl.2012.12.064. Epub 2013 Jan 3.
Gentamicin is an aminoglycoside antibiotic obtained from cultures of Micromonospora as the important anti-infective agents. Gentamicin which lacks 3'-hydroxyl group can avoid the attack from the modification enzymes of antibiotic-resistant bacteria in clinic. Consequently, C-3' dehydroxylation is the key step in gentamicins biosynthesis. We suppose that there are some enzymes responsible for converting intermediate JI-20A to 3',4'-bisdehydroxylated final product gentamicin C(1a), while phosphorylation of 3'-OH is possibly the first step for C-3' dehydroxylation. The gentamicin biosynthetic gene gntI, encoding an aminoglycoside phosphotransferase, was cloned from Micromonospora echinospora ATCC15835 and overexpressed in Escherichia coli. The resulting phosphotransferase was purified, and the kinetic parameters for Kanamycin A, Kanamycin B, Neomycin B and Amikacin were determined. Elucidation of NMR data of phosphorylated kanamycin B has unambiguously demonstrated a regiospecific phosphorylation of 3'-hydroxyl of the 6-aminohexose ring. The results described here partly confirm that the 3'-dehydroxylation step is preceded by a 3' phosphorylation step. It is predicted that GntI belongs to a new aminoglycoside phosphotransferase group involved with aminoglycoside antibiotics biosynthesis pathway.
庆大霉素是一种氨基糖苷类抗生素，从 Micromonospora 的培养物中获得，是重要的抗感染药物。缺乏 3'-羟基的庆大霉素可以避免临床中抗生素耐药菌的修饰酶的攻击。因此，C-3'去羟基化是庆大霉素生物合成的关键步骤。我们假设有一些酶负责将中间产物 JI-20A 转化为 3'、4'-双去羟基的最终产物庆大霉素 C(1a)，而 3'-OH 的磷酸化可能是 C-3'去羟基化的第一步。庆大霉素生物合成基因 gntI 编码氨基糖苷磷酸转移酶，从 Micromonospora echinospora ATCC15835 中克隆并在大肠杆菌中过表达。所得磷酸转移酶被纯化，并确定了卡那霉素 A、卡那霉素 B、新霉素 B 和阿米卡星的动力学参数。对磷酸化卡那霉素 B 的 NMR 数据的阐明明确证明了 6-氨基己糖环 3'-羟基的区域特异性磷酸化。这里描述的结果部分证实了 3'-去羟基化步骤之前是 3'磷酸化步骤。据预测，GntI 属于参与氨基糖苷抗生素生物合成途径的新氨基糖苷磷酸转移酶组。
