结核分枝杆菌铁氧还蛋白还原酶FdrA和FprA在CYP125A1的电子传递链中的作用分析

Analysis of the role of FdrA and FprA in CYP125A1’s electron transfer chain, two ferredoxin reductases in mycobacterium tuberculosis

目的体外评价结核分枝杆菌(Mtb)铁氧还蛋白还原酶FdrA和FprA的活性,探索它们分别与两种铁氧还蛋白的偶联作用,并分析它们在CYP125A1的电子传递链中的作用。方法采用大肠杆菌作为宿主克隆结核分枝杆菌FdrA、FprA和CYP125A1编码基因并进行蛋白外源表达;以NADH或NADPH为电子供体,2,6-二氯酚靛酚(DCPIP)为电子受体评价FdrA及FprA的活性;应用细胞色素C为电子受体研究FdrA或FprA与不同铁氧还蛋白的偶联作用;分析CYP125A1对4-胆甾烯-3-酮的代谢作用进而研究FdrA和FprA在CYP125A1的电子传递链中的作用。结果 FdrA对NADH亲和力较高,Fdx对FdrA活性有明显提升作用,菠菜铁氧还蛋白(spFDX)对其活性没有提升作用,FdrA/Fdx和FdrA/spFDX均不能支持CYP125A1的活性。FprA对NAPDH亲和力较高,Fdx和spFDX均对FprA活性有明显提升作用,Fdx尤甚,FprA/spFDX可以支持CYP125A1的活性,FprA/Fdx不能支持CYP125A1的活性。结论 FprA是结核分枝杆菌CYP125A1的电子传递链蛋白,FdrA可能不是CYP125A1的电子传递链蛋白。

Objective To systematically evaluate the activities of two FDRs （FdrA and FprA）, study the interactions between these two FDRs and two ferredoxins, respectively, and analyze the endogenous redox partners of CYP125A1 in vitro. Methods The cloned genes were ligated to pET-30a（＋） vector and transformed into E.coli BL21 DE3 cells separately. Recombinant proteins were generated by IPTG inducing, followed by affinity purification in Ni column. The activities of FdrA and FprA were evaluated in multilabel reader in the presence of NAD（P）H as the electron donor and DCPIP as the acceptor; the coupling activities of FDR and FDX were analyzed in the presence of cytochrome C as the electron aeceptor; the activity of CYP125A1 which was supported by FdrA or FprA was evaluated by HPLC. Results FdrA preferentially binded NADH and Fdx could increase its activity significantly while spinach ferredoxin （spFDX） didn＇t change its activity. The activity of CYP125A1 couldn＇t be supported by FdrA/Fdx or FdrA/spFDX. FprA preferentially binded NADPH and Fdx or spFDX increased its activity significantly, besides, Fdx had more potent effect. However, only FprA/spFDX could support the activity of CYP 125A 1. Conclusion FprA is one of electron transport chain complex proteins of CYP125A1 and FdrA may not be the redox partner of CYP125A1.