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Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleT JE

DOI: 10.1074/jbc.M116.762336 DOI Help

Authors: Sarah Matthews (University of Manchester) , James D. Belcher (University of Manchester) , Kang Lan Tee (University of Manchester) , Hazel M. Girvan (University of Manchester) , Kirsty J. Mclean (University of Manchester) , Stephen E. J. Rigby (University of Manchester) , Colin W. Levy (University of Manchester) , David Leys (University of Manchester) , David A. Parker (Westhollow Technology Center) , Richard T. Blankley (Agilent Technologies UK Ltd) , Andrew Munro (University of Manchester)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 292 , PAGES 5128 - 5143

State: Published (Approved)
Published: March 2017
Diamond Proposal Number(s): 8997

Open Access Open Access

Abstract: The Jeotgalicoccus sp. 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 OleTJE could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJE active site residues Phe79, His85, and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several per-oxygenases that favor fatty acid hydroxylation. H85Q OleTJE still favors alkene production, suggesting alternative protona- tion 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 His85 in this process. Phe79 interacts with His85, and Phe79 mutants showed diminished affinity for shorter chain (C10–C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245 is cru- cial for binding the substrate carboxylate, and R245E/L muta- tions severely compromise activity and heme content, although alkene products are formed from some substrates, including ste- aric acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

Journal Keywords: crystal structure; cytochrome P450; decarboxylase; enzyme mechanism; fatty acid oxidation; CYP152L1; EPR spectroscopy; alkene; fatty acid hydroxylation; product analysis

Subject Areas: Biology and Bio-materials
Collaborations: Diamond Manchester

Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I24-Microfocus Macromolecular Crystallography

Other Facilities: No

Documents:
5128.full.pdf