Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase

Area of Science:

• Biochemistry
• Enzyme Engineering
• Organic Synthesis

Background:

• Cytochrome P450 enzymes, like CYP102A1 (P450BM3), are versatile monooxygenases used in chemical synthesis.
• Engineering P450BM3 by mutating threonine 268 to glutamate (Thr268Glu) created a peroxygenase variant utilizing hydrogen peroxide (H₂O₂).

Purpose of the Study:

• To evaluate the peroxygenase activity of the Thr268Glu P450BM3 variant.
• To compare its hydroxylation activity and product distribution with the wild-type enzyme.
• To investigate the reaction mechanism using kinetic isotope effects.

Main Methods:

• Site-directed mutagenesis of P450BM3 to create the Thr268Glu variant.
• Enzymatic assays using saturated linear fatty acids and 10-undecenoic acid.
• Kinetic isotope effect studies using deuterated dodecanoic acid.
• Oxidation of tetradecanoic acid using light-driven H₂O₂ generation.

Main Results:

• The Thr268Glu peroxygenase variant exhibited significantly higher peroxide-driven hydroxylation of fatty acids compared to wild-type P450BM3.
• Oxidation occurred predominantly at the ω-1 to ω-3 positions, similar to the wild-type.
• Regioselective allylic hydroxylation at the ω-2 position of 10-undecenoic acid was observed.
• Kinetic isotope effects (7.9–9.5) indicated C-H bond abstraction is crucial, suggesting a compound I intermediate for both systems.

Conclusions:

• The engineered Thr268Glu P450BM3 peroxygenase is a potent catalyst for fatty acid hydroxylation.
• This variant provides a simplified platform for selective cytochrome P450-mediated oxidations.
• The findings support a common reaction mechanism involving compound I in both monooxygenase and peroxygenase modes.