Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences and, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Chembiochem. 2023 Aug 1;24(15):e202300066. doi: 10.1002/cbic.202300066. Epub 2023 Jul 12.
The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.
人们对氧化还原酶催化的利用非天然烟酰胺辅酶的反应越来越感兴趣。完全合成的烟酰胺辅酶仿生（NCB）具有成本效益且易于合成。因此，开发接受 NCB 的酶变得越来越重要。在这里，我们对 SsGDH 进行了工程改造，使其有利于新合成的非天然辅酶 3-氨甲酰-1-（4-羧基苄基）吡啶-1-鎓（BANA）。使用原位配体最小化工具，鉴定出 44 号和 114 号位点为突变热点。所有双突变体的催化活性均提高了 2.7-7.7 倍，最佳双突变体 E44D/E114L 对 BANA 的催化效率提高了 10.6 倍。这些结果为具有多功能 NCB 依赖性的氧化还原酶的合理工程设计以及新型仿生辅酶的设计提供了有价值的信息。
