Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE

Abstract

The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleT_JE (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 OleT_JE 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 OleT_JE 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 peroxygenases that favor fatty acid hydroxylation. H85Q OleT_JE still 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 OleT_JE, 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 OleT_JE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg²⁴⁵ is 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 OleT_JE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

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