Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE

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 peroxygenases that favor fatty acid hydroxylation. H85Q OleTJE 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 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 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 OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry

“…12,62 A number of studies have sought to clarify the active-site requirements that allow OleT to steer the reaction coordinate toward decarboxylation. Particular focus has centered on potential proton donation pathways (e.g., His85) 1,30,63 and hydrogen bonding interactions with solvent 64 for tuning the barrier for oxygen rebound. Although we cannot exclude a possible contribution of these interactions for tuning iron–oxo reactivity, on the basis of the results presented herein, we propose that the position of the fatty acid substrate, bound to key residues in the active site, is critical for steering the reaction at several stages of catalysis.…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…Interestingly the R245L variant gave exclusively alkene product when C18:0 was tested as substrate. The F79A variant of OleT JE mostly decreased the alkene/hydroxy acid product ratio [96].…”

“…The reaction mechanism for the oxidative decarboxylation is under debate [96,97]. After compound I formation by H 2 O 2 via the peroxide shunt, a hydrogen atom is abstracted from the α-or β-position, producing the corresponding carbon radical.…”

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

“…2). It has been known that some P450s can catalyze reactions with the use of H 2 O 2 alone for quite some time (43)(44)(45)(46). Also, mutating residues around the proximal heme ligand allows for alterations in both the heme redox potential and reactivity (47).…”