Abstract: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 acceptor; 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 CYP125A1. Conclusion FprA is one of electron transport chain complex proteins of CYP125A1 and FdrA may not be the redox partner of CYP125A1.
乔峰, 张健美, 白银磊, 杨信怡, 李聪然, 李国庆, 胡辛欣, 游雪甫. 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[J]. 《中国医药生物技术》杂志, 2012, 7(3): 178-186.
QIAO Feng, ZHANG Jian-Mei, BAI Yin-Lei, YANG Xin-Yi, LI Cong-Ran, LI Guo-Qing, HU Xin-Xin, YOU Xue-Fu. Analysis of the role of FdrA and FprA in CYP125A1’s electron transfer chain, two ferredoxin reductases in mycobacterium tuberculosis. , 2012, 7(3): 178-186.