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

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.

Dynamic Control of Electron Transfers in Diflavin Reductases

“…Over the years several additional crystallographic structures were obtained for isolated FMN or FAD domains from BM3 [73,74], the FMN domain of human CPR [75], the flavodoxin-like domain of SiR [76], the isolated FAD domains from nNOS and MSR [77,78]. All these structures superimposed well with the equivalent domains in the structure of the native form of the rat CPR and therefore did not bring any further information about the possible alternative orientations of the domains.…”

Shaking up ancient scents: Insights into santalol synthesis in engineered Escherichia coli

“…Self-sufficient P450 BM3 could be an ideal template for this manipulation because it is readily over-expressed in a soluble form in E. coli and has a higher k cat than any known plant P450 [67,68]. Moreover, the crystal structures of both heme and redox domains have been resolved [69][70][71]. In fact, P450 BM3 from B. megaterium has been successfully engineered to access amorphadiene, specifically yielding artemisinic epoxide [72].…”

NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family

“…In spinach FNR, the homologous residue Tyr314, is displaced by the nicotinamide ring [120, 121]. In NOS isoforms, Phe1395 (nNOS numbering in rat) stacks onto the FAD isoalloxazine ring [96, 104, 122] and, in P450BM3, Trp1046 is found at this position [123], strongly suggesting that these residues are homologs of Trp677 of CYPOR and Tyr414 of FNR. In MSR, Trp697 would be the corresponding residue that modulates hydride transfer from NADPH to FAD and intramolecular ET from FAD to FMN [124].…”