Abstract
Flavocytochrome P450 BM3 is a natural fusion protein constructed of cytochrome P450 and NADPH-cytochrome P450 reductase domains. P450 BM3 binds and oxidizes several mid- to long-chain fatty acids, typically hydroxylating these lipids at the ω-1, ω-2 and ω-3 positions. However, protein engineering has led to variants of this enzyme that are able to bind and oxidize diverse compounds, including steroids, terpenes and various human drugs. The wild-type P450 BM3 enzyme binds inefficiently to many azole antifungal drugs. However, we show that the BM3 A82F/F87V double mutant (DM) variant binds substantially tighter to numerous azole drugs than does the wild-type BM3, and that their binding occurs with more extensive heme spectral shifts indicative of complete binding of several azoles to the BM3 DM heme iron. We report here the first crystal structures of P450 BM3 bound to azole antifungal drugs – with the BM3 DM heme domain bound to the imidazole drugs clotrimazole and tioconazole, and to the triazole drugs fluconazole and voriconazole. This is the first report of any protein structure bound to the azole drug tioconazole, as well as the first example of voriconazole heme iron ligation through a pyrimidine nitrogen from its 5-fluoropyrimidine ring.
Highlights
The cytochromes P450 (P450s or CYPs) are a superfamily of heme b-binding enzymes that catalyze the oxidative modification of a huge number of organic substrates[1]
The weakly bound distal water ligand is readily displaced by a substrate to produce a 5-coordinate high-spin (HS) ferric species that is rapidly reduced to the ferrous state by an electron delivered from the cytochrome P450 reductase (CPR) domain and obtained from NADPH
This leads to dioxygen (O2) binding to heme iron and to the progression of the P450 catalytic cycle that leads to monooxygenation of the substrate[29]
Summary
The cytochromes P450 (P450s or CYPs) are a superfamily of heme b-binding enzymes that catalyze the oxidative modification of a huge number of organic substrates[1]. This is typically achieved through the formation of a highly reactive heme iron-oxo species known as compound I (a ferryl-oxo [FeIV = O] porphyrin radical cation species) that inserts an oxygen atom into a substrate bound close to the heme in the P450 active site[2,3]. There have been a number of successful studies in this area in recent years, including the production of BM3 variants that can bind and hydroxylate propane to propanol, or that catalyze selective carbene transfer from diazoesters to olefins in intact E. coli cells[19,20]. The DM variant appears much more flexible than wild-type (WT) BM3, and can bind and oxidize drug molecules including omeprazole and related gastric proton pump inhibitors (PPIs) to produce human metabolites (e.g. 5-OH esomeprazole, rabeprazole desmethyl ether and lansoprazole sulfone) of these drugs[22,23]
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