Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme

Abstract

The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes

“…Sequence homology analysis suggests that DynA5 adopts a typical class I methyltransferase fold. As the formation of Int-8 does not seem to require a methylation reaction, DynA5 is likely one of the Ado-Met-dependent enzymes that catalyze a nonmethylation reaction. − Considering that epoxidation is known to be catalyzed by flavo- and metalloenzymes, , it will be highly interesting to determine whether and how DynA5 catalyzes the formation of the functionally imperative epoxide group. As neither NADPH nor Ado-Met cofactor is known for their ability to catalyze monooxygenation reactions by activating O 2 , we surmise that the epoxidation may occur via a metabolite or substrate-assisted O 2 activation mechanism that relies on the redox-active anthraquinone to generate a reactive oxygen species such as peroxide .…”

Selective Oxidations Using a Cytochrome P450 Enzyme Variant Driven with Surrogate Oxygen Donors and Light

“…WT CYP199A4 has been demonstrated to catalyse this reaction with high enantioselectivity for the (S)-enantiomer (99 % ee). [38] The T252E peroxygenase enzyme was able to epoxidise 4-vinylbenzoic acid using H 2 O 2 more efficiently than the WT enzyme (Figure 5). The high enantioselectivity for the (S)-enantiomer was also maintained in these peroxygenase reactions (94 % ee, by HPLC Scheme 2).…”

“…Products were identified by co-elution with authentic product standards. [38,44] Products were calibrated against the internal standard. All reactions were performed in triplicate or duplicate.…”

Mechanistic Details of the Sharpless Epoxidation of Allylic Alcohols—A Combined URVA and Local Mode Study

“…The major source of epoxide intermediates is the epoxidation of olefins [17][18][19][20][21][22]. In nature, there exist various enzymes that are capable of oxidizing target substrates with high selectivity to produce epoxides [23][24][25][26].…”