N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis.
N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis.
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DOI：
10.1002/cbic.201100062
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发表时间：
2011-07-25
期刊：
影响因子：
3.2
通讯作者：
Floss, Heinz G.
中科院分区：
文献类型：
--
作者：
Wu, Yingying;Kang, Qianjin;Shang, Guangdong;Spiteller, Peter;Carroll, Brian;Yu, Tin-Wein;Su, Wenjin;Bai, Linquan;Floss, Heinz G.
Ansamitocins是由放线线虫产生的一种有效的抗肿瘤药物。根据它们的结构推断，在各种修饰中，需要在酰胺键上进行N-甲基化。Asm10编码的蛋白属于SAM依赖的甲基转移酶家族。通过基因失活和互补，asm10被证明是导致阿司匹林N-甲基化的原因。凝胶过滤确定Asm10的大小为33.0 kDa，为单体。以N-脱甲基转氨菌素P-3为底物，确定了Asm10催化的最适温度为32℃，最适pH为10.0。ASM10对其他N-脱甲基阿霉菌素和合成吲哚-2-酮也表现出广泛的底物灵活性。通过定点突变，证实Asm10的Asp154和Leu155对其催化作用是必需的，可能是通过与SAM结合。这种独特的N-甲基转移酶的特性丰富了从天然和合成化合物中设计N-甲基化衍生物的工具箱，这将允许对已知的潜在药物进行修饰。
Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The encoded protein by asm10 belongs to SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is 33.0 kDa in size and present as monomer as determined by gel filtration. Using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH were determined to be 32 °C and 10.0 respectively for Asm10 catalysis. Asm10 also showed broad substrate flexibility toward other N-desmethyl ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis possibly thorough the binding of SAM. The characterization of this unique N-methyltransferase enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds, which will allow modification of known potential drugs.
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