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Instruments / Technical Area	Publication Details	Published	Actions
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Novel insights into P450 BM3 interactions with FDA-approved antifungal azole drugs Laura N. Jeffreys , Harshwardhan Poddar , Marina Golovanova , Colin Levy , Hazel M. Girvan , Kirsty J. Mclean , Michael W. Voice , David Leys , Andrew Munro Scientific Reports 9 DOI: 10.1038/s41598-018-37330-y Diamond Proposal Number(s): [17773] Open Access Show Abstract ▼ 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.	Feb 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus Hazel M. Girvan , Harshwardhan Poddar , Kirsty J. Mclean , David R. Nelson , Katherine A. Hollywood , Colin W. Levy , David Leys , Andrew W. Munro Journal Of Inorganic Biochemistry DOI: 10.1016/j.jinorgbio.2018.08.002 Diamond Proposal Number(s): [17773] Open Access Show Abstract ▼ Abstract: The CYP152 family of cytochrome P450 enzymes (P450s or CYPs) is bacterial peroxygenases that use hydrogen peroxide to drive hydroxylation and decarboxylation of fatty acid substrates. We have expressed and purified a novel CYP152 family member – CYP152K6 from the methylotroph Bacillus methanolicus MGA3. CYP152A6 was characterized using spectroscopic, analytical and structural methods. CYP152A6, like its peroxygenase counterpart P450SPα (CYP152B1) from Sphingomonas paucimobilis, does not undergo significant fatty acid-induced perturbation to the heme spectrum, with the exception of a minor Soret shift observed on binding dodecanoic acid. However, CYP152K6 purified from an E. coli expression system was crystallized and its structure was determined to 1.3 Å with tetradecanoic acid bound. No lipids were present in conditions used for crystallogenesis, and thus CYP152K6 must form a complex by incorporating the fatty acid from E. coli cells. Turnover studies with dodecanoic acid revealed several products, with 2-hydroxydodecanoic acid as the major product and much smaller quantities of 3-hydroxydodecanoic acid. Secondary turnover products were undec-1-en-1-ol, 2-hydroxydodec-2-enoic acid and 2,3-dihydroxydodecanoic acid. This is the first report of a 2,3-hydroxylated fatty acid product made by a peroxygenase P450, with the dihydroxylated product formed by CYP152K6-catalyzed 3-hydroxylation of 2-hydroxydodecanoic acid, but not by 2-hydroxylation of 3-hydroxydodecanoic acid.	Aug 2018

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