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Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus

DOI: 10.1016/j.jinorgbio.2018.08.002 DOI Help

Authors: Hazel M. Girvan (The University of Manchester; University of Tennessee Health Science Center) , Harshwardhan Poddar (Manchester Institute of Biotechnology, The University of Manchester) , Kirsty J. Mclean (Manchester Institute of Biotechnology, The University of Manchester) , David R. Nelson (University of Tennessee Health Science Center) , Katherine A. Hollywood (Manchester Institute of Biotechnology, The University of Manchester) , Colin W. Levy (Manchester Institute of Biotechnology, The University of Manchester) , David Leys (Manchester Institute of Biotechnology, The University of Manchester) , Andrew W. Munro (Manchester Institute of Biotechnology, The University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Inorganic Biochemistry

State: Published (Approved)
Published: August 2018
Diamond Proposal Number(s): 17773

Open Access Open Access

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.

Journal Keywords: Peroxygenase; Cytochrome P450; Organic products; Protein structure; Substrate binding; EPR spectroscopy

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Documents:
1-s2.0-S0162013418301624-main.pdf

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