4DQL

Crystal structure of the FAD binding domain of cytochrome P450 BM3 in complex with NADP+

4DQL の概要

• エントリーDOI: 10.2210/pdb4dql/pdb
• 関連するPDBエントリー: 4DQK
• 分子名称: Bifunctional P-450/NADPH-P450 reductase, FLAVIN-ADENINE DINUCLEOTIDE, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ... (7 entities in total)
• 機能のキーワード: rossmann fold, redox, fad and nadp+ binding, oxidoreductase
• 由来する生物種: Bacillus megaterium
• 細胞内の位置: Cytoplasm (By similarity): P14779
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 92134.61

構造登録者

joyce, M.G.,leys, D. (登録日: 2012-02-16, 公開日: 2012-03-07, 最終更新日: 2024-02-28)

主引用文献

Joyce, M.G.,Ekanem, I.S.,Roitel, O.,Dunford, A.J.,Neeli, R.,Girvan, H.M.,Baker, G.J.,Curtis, R.A.,Munro, A.W.,Leys, D.
The crystal structure of the FAD/NADPH-binding domain of flavocytochrome P450 BM3.
Febs J., 279:1694-1706, 2012

PubMed Abstract: 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.

PubMed: 22356131
DOI: 10.1111/j.1742-4658.2012.08544.x

実験手法

X-RAY DIFFRACTION (2.15 Å)