Institute for Biochemistry, Biotechnology and Bioinformatics, Technical University Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
Acta Crystallogr F Struct Biol Commun. 2020 Aug 1;76(Pt 8):334-340. doi: 10.1107/S2053230X2000922X. Epub 2020 Jul 28.
The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.
热纤梭菌的铁氧还蛋白还原酶 FdR9 属于与加氧酶偶联的 NADH 依赖型铁氧还蛋白还原酶 (FNR) 家族成员，可催化电子从 NADH 转移至其生理电子受体铁氧还蛋白。它是热纤梭菌中假定的三组分细胞色素 P450 单加氧酶系统的一部分，该系统由 CYP222A1 和 [3Fe-4S]-簇铁氧还蛋白 Fdx8 以及 FdR9 组成。在此，FdR9 被过表达和纯化，并以 1.9 Å 的分辨率确定其晶体结构。FdR9 的整体结构与 FNR 家族的其他成员相似，由 FAD 结合域、NAD 结合域和 C 末端域组成。用 FdR9 进行的活性测量证实其强烈偏好 NADH 作为辅助因子。将 FdR9 的 FAD 和 NAD 结合域与其他铁氧还蛋白还原酶的结合域进行比较，发现 FAD 结合域中存在保守的序列基序，以及几个涉及 FAD 和 NAD 辅助因子结合的高度保守残基。此外，FdR9 的 NAD 结合位点包含一个修饰的 Rossmann 折叠基序 GxSxxS，而不是经典的 GxGxxG 基序。
