Cieślak Jolanta, Miyanaga Akimasa, Takaku Ryoma, Takaishi Makoto, Amagai Keita, Kudo Fumitaka, Eguchi Tadashi
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Proteins. 2017 Jul;85(7):1238-1247. doi: 10.1002/prot.25284. Epub 2017 Mar 29.
Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β-amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand-alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β-amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3-aminobutanoic acid and 3-aminononanoic acid, respectively. We solved the X-ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β-amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β-amino group of the substrates. However, the hydrophobic side-chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3-aminobutanoate of the trapped acyl-adenylate intermediate to construct a shallow substrate-binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3-aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β-amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238-1247. © 2017 Wiley Periodicals, Inc.
诸如因塞德宁和克雷米霉素之类的大环内酰胺抗生素具有脂肪族β-氨基酸作为其聚酮链的起始单元。在因塞德宁和克雷米霉素的生物合成中，独特的独立腺苷化酶IdnL1和CmiS6选择并激活合适的脂肪族β-氨基酸作为起始单元。在本研究中，我们描述了IdnL1和CmiS6的酶学特性及底物特异性的结构基础。功能分析表明，IdnL1和CmiS6分别识别3-氨基丁酸和3-氨基壬酸。我们解析了IdnL1和CmiS6的X射线晶体结构，以了解这些脂肪族β-氨基酸的识别机制。这些结构表明，IdnL1和CmiS6共享一个与底物的β-氨基相互作用的共同识别基序。然而，两种酶中底物的疏水侧链容纳方式不同。IdnL1在靠近捕获的酰基-腺苷酸中间体的3-氨基丁酸酯末端甲基处有一个大的Leu220，以构建一个浅的底物结合口袋。相反，CmiS6在相应位置具有Gly220以容纳3-氨基壬酸。这一结构观察结果得到了突变研究的支持。因此，220位氨基酸残基的大小对于这些腺苷化酶中脂肪族β-氨基酸底物的选择至关重要。《蛋白质》2017年；85:1238 - 1247。©2017威利期刊公司。
