4dql
Crystal structure of the FAD binding domain of cytochrome P450 BM3 in complex with NADP+Crystal structure of the FAD binding domain of cytochrome P450 BM3 in complex with NADP+
Structural highlights
Function[CPXB_BACME] Functions as a fatty acid monooxygenase. Catalyzes hydroxylation of medium and long-chain fatty acids at omega-1, omega-2 and omega-3 positions, with optimum chain lengths of 12-16 carbons (lauric, myristic, and palmitic acids). The reductase domain is required for electron transfer from NADP to cytochrome P450. Publication Abstract from PubMedWe report the crystal structure of the FAD/NADPH-binding domain of the biotechnologically important flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both absence and presence of the ligand NADP(+) , identifying important protein interactions with the NADPH 2'-phosphate that help 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. Multi-angle 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. In 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/NADPH domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin-like) domain within the BM3 reductase module. The crystal structure of the FAD/NADPH binding domain of flavocytochrome P450 BM3.,Gordon Joyce M, Ekanem IS, Roitel O, Dunford AJ, Neeli R, Girvan HM, Baker GJ, Curtis RA, Munro AW, Leys D FEBS J. 2012 Feb 22. doi: 10.1111/j.1742-4658.2012.08544.x. PMID:22356131[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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