title: Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE.
abstract: TheJeotgalicoccussp. peroxygenase cytochrome P450 OleTJE(CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJEcould enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJEactive site residues Phe79, His85, and Arg245to interrogate their roles in substrate binding and catalytic activity. His85is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleTJEstill favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-induced heme iron spin state shift toward high spin by comparison with WT OleTJE, indicating the key role of His85in this process. Phe79interacts with His85, and Phe79mutants showed diminished affinity for shorter chain (C10-C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJEis least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245is crucial for binding the substrate carboxylate, and R245E/L mutations severely compromise activity and heme content, although alkene products are formed from some substrates, including stearic acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleTJEcatalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Matthews
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: James D
authors.initials: JD
authors.lastname: Belcher
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Kang Lan
authors.initials: KL
authors.lastname: Tee
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Girvan
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: Colin W
authors.initials: CW
authors.lastname: Levy
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.affiliation: the Westhollow Technology Center, Houston, Texas 77028-3101, and.
authors.forename: David A
authors.initials: DA
authors.lastname: Parker
authors.affiliation: Agilent Technologies UK Ltd., Cheshire SK8 3GR, United Kingdom.
authors.forename: Richard T
authors.initials: RT
authors.lastname: Blankley
authors.affiliation: From the Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom, Andrew.Munro@Manchester.ac.uk.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
mesh_terms: D000475:Alkenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D006900:Hydroxylation; D008958:Models, Molecular; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D040141:Staphylococcaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000475:Alkenes; D005227:Fatty Acids; D003577:Cytochrome P-450 Enzyme System; D010544:Peroxidases; C066418:cytochrome P-450 peroxygenase
keywords: CYP152L1; EPR spectroscopy; alkene; crystal structure; cytochrome P450; decarboxylase; enzyme mechanism; fatty acid hydroxylation; fatty acid oxidation; product analysis