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

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.

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。

前期在麦草畏降解菌 Rhizorhabdus dicambivorans Ndbn-20 中鉴定了1个四氢叶酸（THF）依赖型麦草畏脱甲基基因簇，包括一个THF依赖型脱甲基酶基因dmt66 和三个四氢叶酸代谢相关基因（mthfr66, purU和dhch）。本项目目标是研究Mthfr66的功能、酶学特性和结构，揭示Mthfr66在THF依赖型脱甲基酶系统辅酶THF再生中的作用。对Mthfr66进行了外源表达和纯化。研究了Mthfr66酶学特性和催化动力学。将Mthfr66的5个半胱氨酸均突变为丙氨酸，构建了具有更高稳定性突变体Mthfr66m，解析了Se-Mthfr66m晶体的三维结构。Mthfr66m是由两个相同亚基组成的同源二聚体，单体由15个α-helix和10个β-strand组成，催化中心是由8个α-helix和8个β-strand组成的桶状结构。通过分子对接推测了FAD和5-CH3-THF与Mthfr66的结合位点。获得15个Mthfr66定点突变体，其中5个突变体活性几乎完全丧失。敲除mthfr66基因导致突变体Ndbn-20∆metF1完全失去转化5-CH3-THF和降解麦草畏能力，表明Mthfr66是Ndbn-20细胞内唯一有活性的5-CH3-THF脱氢酶。Mthfr66生理功能是催化将麦草畏脱甲基生成的5-CH3-THF转化为5,10-CH2-THF（进而自发或酶催化生成THF），从而使辅酶THF得到再生，使麦草畏脱甲基反应能循环进行。构建了dmt50+mthfr66共转基因拟南芥，共转的mthfr66基因可以解除5-CH3-THF对脱甲基酶Dmt50的反馈抑制，从而使共转基因拟南芥对麦草畏耐受性显著高于dmt50转基因拟南芥和非转基因拟南芥。本项目从生理、生化、遗传和蛋白结构角度揭示了Mthfr66 的结构、功能及其在THF依赖型脱甲基酶系统中THF再生中的作用，研究成果对深入研究四氢叶酸依赖型麦草畏脱甲基酶系统的分子机制以及利用这一系统构建耐受高浓度麦草畏转基因作物提供理论依据。