双功能酶Mthfr66的特性及其在四氢叶酸依赖型麦草畏脱甲基途径中的生理功能研究

脱甲基是甲基取代芳烃化合物微生物降解的重要步骤，四氢叶酸依赖型脱甲基是主要的脱甲基途径之一，但目前对该途径的机制特别是四氢叶酸再生机制还不清楚。前期在麦草畏降解菌株Ndbn-20鉴定了1个麦草畏脱甲基基因簇，它包括一个四氢叶酸依赖型脱甲基酶基因和三个四氢叶酸代谢相关基因，其中mthfr66表达产物体外具有5-甲基四氢叶酸水解酶和5,10-亚甲基四氢叶酸还原酶两种活性。本项目研究Mthfr66两种催化活性的特性和动力学，筛选Mthfr66失去水解酶或还原酶活性的突变体，初步解析Mthfr66晶体结构，研究mthfr66突变及敲除对Ndbn-20转化麦草畏速率的影响，揭示Mthfr66两种催化活性的特性及其在菌株Ndbn-20麦草畏脱甲基过程中的生理功能。预期结果为深入研究Mthfr66的结构和催化机制及四氢叶酸依赖型脱甲基途径中四氢叶酸再生机制提供理论依据。

Demethylation is an important step in the microbial degradation of methyl-substituted aromatic compounds. Tetrahydrofolate (H4folate)-dependent demethylation is one of the main demethylation pathways. However, the mechanism of this pathway, especially the regeneration of tetrahydrofolate, is unclear. Previously, Dicamba-degrading strain Ndbn-20 was isolated from compost. The dicamba demethylation is via a H4folate-dependent demethylases system in the strain, and a H4folate-dependent dicamba demethylation gene cluster was identified. This gene cluster consists of a dicamba methyltransferase gene dmt66 and three genes (methfr66, dhch, and purU) related to the metabolism of tetrahydrofolate. Mthfr66 is a bifunctional enzyme (5,10-CH2-H4folate reductase/5-CH3-H4folate hydrolase) in vitro. This study was designed to investigate the enzymatic characteristics and kinetics of Mthfr66, to screen Mthfr66 mutants that lost 5,10-CH2-H4folate reductase or 5-CH3-H4folate hydrolase activity, preliminary analyze the structure of Mthfr66, and to study the effect of mutation and knock out of methfr66 on the conversion efficiencies of dicamba by stain Ndbn-20. This project will reveal the enzymatic characteristics of Mthfr66 and its role in the dicamba demethylation process in vivo, and provide theoretical basis for further studies of the structure and catalytic mechanism of Mthfr66 and the regeneration mechanism of H4folate in H4folate-dependent demethylation pathway.