N-脱甲基酶电子转移受体蛋白CdnD及其Rieske型[2Fe-2S]结构域的功能表征  

Functional characterization of N-demethylase electrons transfer receptor protein CdnD and its Rieske type[2Fe-2S]domain

作　　者：李会佳 燕云 孟亦轲 高子晴[1] LI Huijia;YAN Yun;MENG Yike;GAO Ziqing(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2025年第2期109-114,共6页Journal of Dalian Polytechnic University

基　　金：辽宁省民族药功效成分开发与应用重点实验室开放课题(2021JH13/10200038)。

摘　　要：通过对咖啡因降解菌Paraburkholderia caffeinilytica CF1中咖啡因降解相关基因簇上的N-脱甲基酶电子转移受体蛋白基因cdnD对应的CdnD蛋白进行异源表达、纯化及酶活力测定,确定其功能。对CdnD酶性质研究发现,其较同家族其他氧化还原酶多了一个Rieske型[2Fe-2S]结构域。为研究这一结构域在N-脱甲基过程中的功能,敲除了CdnD的Rieske型[2Fe-2S]结构域,构建敲除突变株CdnDΔD1,并研究了其对N_(1)-脱甲基酶CdnA与N_(7)-脱甲基酶CdnC活性的影响。结果表明,CdnDΔD1与CdnA共同作用可脱去底物的N_(1)-甲基,而与CdnC和CdnE协同作用不能脱去底物的N_(7)-甲基,因此确定CdnD的Rieske型[2Fe-2S]结构域在N_(7)-脱甲基过程中起到电子传递结构域的作用。A gene named cdnD from caffeine degrading bacterium Paraburkholderia caffeinelytica CF1 is on the caffeine degradation related gene cluster and corresponding to the N-demethylase electron transfer receptor protein.Its function was determined by heterologous expression,purification and enzyme activity The function of CdnD was determined by heterologous expression,purification and enzyme activities.It has been found that there is an additional Rieske type[2Fe-2S]domain in CdnD compared to other oxidoreductases in the same family.To investigate the role of the domain in the N-demethylation process,the Rieske type[2Fe-2S]domain of CdnD was knocked out and the mutant strain CdnDΔD1 was constructed.Its effects on the activities of N_(1)-demethylase CdnA and N_(7)-demethylase CdnC were studied respectively.The results showed that the N_(1)methyl group of the substrate could be removed by CdnDΔD1 combined with CdnA,but the N_(7)methyl group of the substrate could not be removed by CdnDΔD1 combined with CdnC and CdeE.Therefore,it has been proved that the electrons could be transported by the Rieske type[2Fe-2S]domain of CdnD in the catalytic process of N_(7)-demethylation.

关 键 词：咖啡因 N-脱甲基酶 Rieske型[2Fe-2S] 电子转移受体蛋白 

分 类 号：Q71[生物学—分子生物学]