Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from .

The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus and its heme (P450) domain were expressed in and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid ( = 2.7 μM).

The crystal structure of the FAD/NADPH-binding domain of flavocytochrome P450 BM3.

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.