菌株Sphingobium sp. Na-1代谢除草剂敌草胺手性异构体的分子机制

Pesticides with chiral isomers occupy an important position in the global pesticide market. As the different isomers of chiral pesticides show different biological activities and ecotoxicological activities, the dynamics of the disappearance of different isomers in the environment are of great concern. Napropamide is an important herbicide belonging to the amide herbicides. However, its microbial degradation and transformation, especially the molecular catabolic mechanism involved in the catabolism of different chiral isomers, largely remains unknown. In this project, on the basis of a previously isolated strain of Sphingobium sp. Na-1, which can degrade both chiral isomers (S-Napropamide and R-Napropamide), while in a different metabolic pathway, we will clone the dioxygenase genes responsible for the initial degradation of both chiral isomers of napropamide; elucidate the catalytic mechanism of the identified dioxygenase as well as the mechanism underlying the different dioxygenation positions for different chiral isomers; reveal the complete metabolic pathway of S-napropamide as well as the key genes involved in the downstream pathway of S-napropamide. We will fully elucidate the catabolic mechanism of two chiral isomers of napropamide in the molecular level in strain Na-1. The outcomes of this project will not only enrich our theory of microbial metabolism of chiral pesticides, provide technical guidance for the risk assessment, safe application and bioremediation of chiral pesticides, but also provide theoretical guidance for the rational design of specific enzymes for pesticide chiral separation.

具有手性异构体的农药在全球农药市场中占有重要地位。手性农药的不同异构体通常具有不同的生物活性和生态毒理，因此，其异构体在环境中的消亡动态成为关注的热点。敌草胺作为一类重要的酰胺类除草剂，然而其微生物参与的代谢转化、尤其是其不同手性异构体的代谢机制研究却十分滞后。本项目将以分离获得的一株能同时降解S-和R-敌草胺两种手性异构体、但降解方式不同的菌株Sphingobium sp. Na-1为研究对象，克隆起始降解敌草胺手性异构体的双加氧酶基因，阐明双加氧酶的催化机制以及对不同手性底物双加氧位置发生差异的机制，揭示S-敌草胺的完整代谢途径和下游途径关键酶基因，最终阐明菌株Na-1代谢敌草胺手性异构体的分子机制。项目成果不仅能丰富手性农药的微生物代谢机制理论，为手性农药环境污染风险评估、安全使用及微生物修复提供技术指导，甚至能为实现敌草胺手性拆分特定酶的改造提供理论指导。

具有手性异构体的农药在全球农药市场中占有重要地位。手性农药的不同异构体通常具有不同的生物活性和生态毒理，因此，其异构体在环境中的消亡动态成为关注的热点。敌草胺作为一类重要的酰胺类除草剂，然而其微生物参与的代谢转化、尤其是其不同手性异构体的代谢机制研究却十分滞后。本项目从生产敌草胺的农药厂土壤里筛选分离到两株敌草胺高效降解菌株Sphingobium sp. strain A1和Sphingobium sp. strain B2，发现菌株A1和菌株B2对R-/S-敌草胺的降解能力和降解途径均存在较大差异，揭示了菌株Sphingobium sp. A1和B2中R-/S-敌草胺两种手性异构体的完整代谢途径；克隆到催化敌草胺酰胺键断裂的酰胺酶基因snaH，揭示了SnaH对R-敌草胺（R-NAP）和R-敌草胺（S-NAP）手性选择性机制；S构型Zn配位O原子与羰基C原子距离较近，O原子的电子容易进攻C核，从而形成反应步骤中的过渡态复合物；而R构型的配体分子因位阻而远离W72和配位O原子，从而妨碍了过渡态复合物的形成。克隆到催化S-NAP 产物S-2-(1-萘氧基)丙酸（S-NP）醚键断裂的双加氧酶基因snpd，揭示了Snpd专一性催化S-NP的手性特异性机制，Snpd与S-NP的结合力显著强于R-NP，且S-NP手性中心的H原子与Fe配体O=原子距离更近。项目成果不仅能丰富手性农药的微生物代谢机制理论，为手性农药环境污染风险评估、安全使用及微生物修复提供技术指导，甚至能为实现敌草胺手性拆分特定酶的改造提供理论指导。