School of Biosciences, University of Birmingham, UK.
School of Science, RMIT University, Melbourne, Australia.
FEBS Lett. 2022 Sep;596(18):2425-2440. doi: 10.1002/1873-3468.14413. Epub 2022 Jul 13.
Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.
硝基还原酶激活硝基芳香族抗生素和癌症前体药物，生成细胞毒性羟胺，并将醌还原为氢醌。我们通过稳态和停流动力学研究表明，大肠杆菌硝基还原酶 NfsA 利用 NADPH 的活性比利用 NADH 的活性高 20-50 倍，且产物释放可能是限速步骤。NfsA 与 NADP 的晶体结构显示，一个可移动的环形成了一个磷酸盐结合口袋。烟酰胺环和烟酰胺核糖是可移动的，这在分子动力学（MD）模拟中得到了证实。我们提出了 NADPH 与 NfsA 结合的模型。只看到一个 NADP 结合到 NfsA 二聚体上，而 MD 模拟表明结合第二个 NADP（H）辅助因子是不利的，这表明 NfsA 和该蛋白质超家族的其他成员可能具有半位点机制。
