Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

Human P450-like oxidation of diverse proton pump inhibitor drugs by 'gatekeeper' mutants of flavocytochrome P450 BM3.

Production of drug metabolites is one area where enzymatic conversion has significant advantages over synthetic chemistry. These high value products are complex to synthesize, but are increasingly important in drug safety testing. The vast majority of drugs are metabolized by cytochromes P450 (P450s), with oxidative transformations usually being highly regio- and stereo-selective. The PPIs (proton pump inhibitors) are drugs that are extensively metabolized by human P450s, producing diverse metabolites dependent on the specific substrate. In the present paper we show that single mutations (A82F and F87V) in the biotechnologically important Bacillus megaterium P450 BM3 enzyme cause major alterations in its substrate selectivity such that a set of PPI molecules become good substrates in these point mutants and in the F87V/A82F double mutant. The substrate specificity switch is analysed by drug binding, enzyme kinetics and organic product analysis to confirm new activities, and X-ray crystallography provides a structural basis for the binding of esomeprazole to the F87V/A82F enzyme. These studies confirm that such 'gatekeeper' mutations in P450 BM3 produce major perturbations to its conformation and substrate selectivity, enabling novel P450 BM3 reactions typical of those performed by human P450s. Efficient transformation of several PPI drugs to human-like products by BM3 variants provides new routes to production of these metabolites.

Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias.

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such "gatekeeper" mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme.

Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: implications for the mechanism of electron transfer in the P450 BM3 dimer.

Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic "shield" from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K(m) for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.

Novel insights into P450 BM3 interactions with FDA-approved antifungal azole drugs.

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.