Zhu Yuhao, Mori Takahiro, Karasawa Masayuki, Shirai Kohei, Cheng Wenjiao, Terada Tohru, Awakawa Takayoshi, Abe Ikuro
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan.
Nat Commun. 2024 Dec 30;15(1):10896. doi: 10.1038/s41467-024-55265-z.
The general control non-repressible 5 (GCN5)-related N-acetyltransferase (GNAT) SbzI, in the biosynthesis of the sulfonamide antibiotic altemicidin, catalyzes the transfer of the 2-sulfamoylacetyl (2-SA) moiety onto 6-azatetrahydroindane dinucleotide. While most GNAT superfamily utilize acyl-coenzyme A (acyl-CoA) as substrates, SbzI recognizes a carrier-protein (CP)-tethered 2-SA substrate. Moreover, SbzI is the only naturally occurring enzyme that catalyzes the direct incorporation of sulfonamide, a valuable pharmacophore in medicinal chemistry. Here, we present the structure-function analysis and structure-based engineering of SbzI. The crystal structure of SbzI in complex with the CP SbzG, along with cross-linking and isothermal titration calorimetry analyses of their variants, revealed the structural basis for CP recognition by the GNAT SbzI. Furthermore, docking simulation, molecular dynamics simulation, and mutagenesis studies indicated the intimate structural details of the unique reaction mechanism of SbzI, which does not utilize a general base residue in contrast to other typical GNATs. These findings facilitated rational engineering of the enzyme to expand the substrate range and to generate azaindane dinucleotide derivatives.
在磺酰胺类抗生素阿替米星的生物合成中，与一般控制非阻遏蛋白5（GCN5）相关的N - 乙酰基转移酶（GNAT）SbzI催化将2 - 氨磺酰乙酰（2 - SA）部分转移到6 - 氮杂四氢茚二核苷酸上。虽然大多数GNAT超家族利用酰基辅酶A（acyl - CoA）作为底物，但SbzI识别与载体蛋白（CP）相连的2 - SA底物。此外，SbzI是唯一一种天然存在的催化直接掺入磺酰胺的酶，磺酰胺是药物化学中有价值的药效基团。在此，我们展示了SbzI的结构 - 功能分析和基于结构的工程改造。SbzI与CP SbzG复合物的晶体结构，以及它们变体的交联和等温滴定量热分析，揭示了GNAT SbzI识别CP的结构基础。此外，对接模拟、分子动力学模拟和诱变研究表明了SbzI独特反应机制的详细结构细节，与其他典型的GNATs不同，SbzI不利用一般的碱基残基。这些发现有助于对该酶进行合理的工程改造，以扩大底物范围并生成氮杂茚二核苷酸衍生物。
