Department of Microbiology, Institute of Biomedical Science , University of São Paulo , Avenida Prof. Lineu Prestes 1374 , 05508-900 São Paulo , Brazil.
Department of Pharmacy, Faculty of Pharmaceutical Sciences , University of São Paulo , Prof. Lineu Prestes Avenue 580 , 05508-900 São Paulo , Brazil.
ACS Chem Biol. 2019 May 17;14(5):925-933. doi: 10.1021/acschembio.9b00115. Epub 2019 Apr 30.
Gentamicins are clinically relevant aminoglycoside antibiotics produced by several Micromonospora species. Gentamicins are highly methylated and functionalized molecules, and their biosynthesis include glycosyltransferases, dehydratase/oxidoreductases, aminotransferases, and methyltransferases. The biosynthesis of gentamicin A from gentamicin A2 involves three enzymatic steps that modify the hydroxyl group at position 3″ of the unusual garosamine sugar to provide its substitution for an amino group, followed by an N-methylation. The first of these reactions is catalyzed by GenD2, an oxidoreductase from the Gfo/Idh/MocA protein family, which reduces the hydroxyl at the C3″ of gentamicin A to produce 3''-dehydro-3''-oxo-gentamicin A2 (DOA2). In this work, we solved the structure of GenD2 in complex with NAD+. Although the structure of GenD2 has a similar fold to other members of the Gfo/Idh/MocA family, this enzyme has several new features, including a 3D-domain swapping of two β-strands that are involved in a novel oligomerization interface for this protein family. In addition, the active site of this enzyme also has several specialties which are possibly involved in the substrate specificity, including a number of aromatic residues and a negatively charged region, which is complementary to the polycationic aminoglycoside-substrate. Therefore, docking simulations provided insights into the recognition of gentamicin A2 and into the catalytic mechanism of GenD2. This is the first report describing the structure of an oxidoreductase involved in aminoglycoside biosynthesis and could open perspectives into producing new aminoglycoside derivatives by protein engineering.
庆大霉素是由几种 Micromonospora 物种产生的临床相关氨基糖苷类抗生素。庆大霉素是高度甲基化和功能化的分子，其生物合成包括糖基转移酶、脱水酶/氧化还原酶、氨基转移酶和甲基转移酶。从庆大霉素 A2 生物合成庆大霉素 A 涉及三个酶促步骤，这些步骤修饰了异常 garosamine 糖在位置 3″的羟基，为其提供了氨基取代，然后进行 N-甲基化。这些反应中的第一个由 GenD2 催化，GenD2 是 Gfo/Idh/MocA 蛋白家族的氧化还原酶，它将庆大霉素 A 的 C3″上的羟基还原为 3''-去氢-3''-氧-庆大霉素 A2 (DOA2)。在这项工作中，我们解决了 GenD2 与 NAD+复合物的结构。尽管 GenD2 的结构与 Gfo/Idh/MocA 家族的其他成员具有相似的折叠，但该酶具有几个新的特征，包括两个涉及该蛋白家族新的寡聚化界面的β-链的 3D 结构域交换。此外，该酶的活性位点还具有几个可能与底物特异性相关的特征，包括一些芳香族残基和一个带负电荷的区域，该区域与多阳离子氨基糖苷类底物互补。因此，对接模拟提供了对庆大霉素 A2 识别和 GenD2 催化机制的深入了解。这是第一个描述参与氨基糖苷类生物合成的氧化还原酶结构的报告，可能为通过蛋白质工程生产新的氨基糖苷类衍生物开辟了前景。
