Structural Basis of the Selectivity of GenN, an Aminoglycoside N-Methyltransferase Involved in Gentamicin Biosynthesis
Structural Basis of the Selectivity of GenN, an Aminoglycoside N-Methyltransferase Involved in Gentamicin Biosynthesis
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GenN（一种参与庆大霉素生物合成的氨基糖苷N-甲基转移酶）选择性的结构基础
DOI：
10.1021/acschembio.7b00466
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发表时间：
2017-11-01
影响因子：
4
通讯作者：
Bertacine Dias, Marcio Vinicius
中科院分区：
文献类型：
--
作者：
Bury, Priscila dos Santos;Huang, Fanglu;Bertacine Dias, Marcio Vinicius
庆大霉素是由棘孢小单孢菌产生的高度甲基化的、具有临床价值的假三糖抗生素。GenN已被表征为与其他酶具有低序列相似性的S-腺苷-L-甲硫氨酸依赖性甲基转移酶。它是负责3？- 3“-氨基-3”-氨基-庆大霉素A2的N-甲基化，是庆大霉素C复合物生物合成途径中环III的一种重要修饰。纯化的重组GenN还有效催化相关氨基糖苷类卡那霉素B和妥布霉素的3“-N-甲基化，这两种药物在环III的C5 "位置都含有额外的羟甲基。我们已经获得了8个共晶结构的GenN，在2.2埃或更好的分辨率，包括二元复合物的GenN和S-腺苷-L-同型半胱氨酸（SAH）和三元复合物的GenN，SAH，和几个氨基糖苷类。GenN结构揭示了在任何其他N-甲基转移酶中未观察到的几个特征，这些特征适合其在庆大霉素生物合成中的作用。这些包括一个新的N-末端结构域，可能参与蛋白质：蛋白质与庆大霉素X2生物合成的上游酶的相互作用，以及两个参与氨基糖苷类底物识别的长环。此外，与不同的配体的复杂的GenN的结构的分析，支持的结果，活性位点诱变，使我们能够提出一种催化机制，并揭示了结构基础的惊人能力的天然GenN对这些替代基板。
Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3?-N-methylation of 3 ''-dehydro-3 ''-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3 ''-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5 '' position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 angstrom or better, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.