Department of Biochemistry, University of Leicester, Leicester, UK.
FEBS J. 2012 May;279(9):1694-706. doi: 10.1111/j.1742-4658.2012.08544.x. Epub 2012 Mar 16.
We report the crystal structure of the FAD/NADPH-binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP(+), identifying important protein interactions with the NADPH 2'-phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH-dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild-type FAD domain to be near-exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme's connecting domain (that links the FAD domain to the FMN-binding domain in P450 BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin-like) domain within the BM3 reductase module.
我们报告了生物技术上重要的巨大芽孢杆菌黄素细胞色素 P450 BM3 的 FAD/NADPH 结合域（FAD 结构域）的晶体结构，这是该酶最后一个解析结构的结构域。该结构在没有和存在配体 NADP(+)的情况下均得到了确定，鉴定了与 NADPH 2'-磷酸的重要蛋白相互作用，有助于决定对 NADPH 的特异性而不是 NADH，涉及残基 Tyr974、Arg966、Lys972 和 Ser965。Trp1046 侧链将 FAD 异咯嗪环与 NADPH 隔开，该残基的运动对于使 FAD 依赖于 NADPH 的还原是必需的。多种结合相互作用稳定 FAD 辅因子，包括与 Tyr860 和 Trp854 侧链的腺嘌呤基团的芳香堆积，以及与 FAD 焦磷酸氧的几个相互作用，包括与 Tyr828、829 和 860 的键合。C773 和 C999 突变为丙氨酸是成功结晶所必需的，C773A 预测不利于分子内和分子间二硫键形成。多角度激光散射分析表明，野生型 FAD 结构域几乎完全是二聚体，用还原剂二硫苏糖醇处理可破坏二聚体。相比之下，光散射表明 C773A/C999A FAD 结构域是单体。C773A/C999A FAD 结构域结构证实 Ala773 是表面暴露的，并且与 Cys810 接近，该酶的连接结构域（将 FAD 结构域与 P450 BM3 中的 FMN 结合结构域连接）的这一区域位于 FAD 结构域之间的晶体接触界面处。FAD 结构域的晶体结构能够对其与 BM3 还原酶模块中的同源 FMN（黄素蛋白样）结构域的相互作用进行分子建模。
