Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7 mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.

中文翻译：

---

结合异戊二烯基转移酶NovQ在Glu281上的诱变和代谢工程策略以提高对甲萘醌的烯丙基化活性。

异戊二烯基转移酶NovQ是至关重要的一类，涉及次生代谢物如氯霉素和新霉素的生物合成。为了研究NovQ的结构与催化性能之间的关系，在这里，我们分析了底物结合位点，即PT桶，并发现甲萘醌对苯二酚与活性口袋中心附近的残基Glu281形成了分子间相互作用。在这项研究中，Glu281被9种不同的氨基酸取代，并且在体外观察到了突变体的催化特性。其中，E281Q对芳香族底物和异戊二烯基供体的活性为2.05倍，而其他的催化效率为野生型NovQ的8.4％至88.6％。此外，考虑了催化条件和底物状态对NovQ及其突变体活性的影响，以得到优化的异戊二烯化反应。当进化的NovQ变体E281Q在通过工程戊酸（MVA）途径合成二磷酸二甲基烯丙酯的宿主中过表达时，我们通过外源提供芳香族底物在C-3位置收获了4.7 mg / L的烯丙基甲萘醌。基于NovQ的微生物平台的构建为在大肠杆菌中进一步生物合成各种维生素K2与其他ABBA异戊二烯基转移酶开辟了新的方向。通过外源供应芳香族底物，在C-3位置处7 mg / L戊烯基甲萘醌。基于NovQ的微生物平台的构建为在大肠杆菌中进一步生物合成各种维生素K2与其他ABBA异戊二烯基转移酶开辟了新的方向。通过外源提供芳香族底物，在C-3位置处7 mg / L戊烯基甲萘醌。基于NovQ的微生物平台的构建为在大肠杆菌中进一步生物合成各种维生素K2与其他ABBA异戊二烯基转移酶开辟了新的方向。