Temperature-dependent conformational dynamics govern regioselectivity in a CYP152 decarboxylase

Area of Science:

• Biocatalysis and Enzyme Engineering
• Chemical Biology
• Structural Biology

Background:

• Peroxygenases from the CYP152 family catalyze fatty acid oxidation using hydrogen peroxide, bypassing traditional redox partners.
• A subset of these enzymes decarboxylates fatty acids into terminal alkenes, crucial for renewable fuels and petrochemicals.
• Understanding the mechanism controlling decarboxylation versus hydroxylation selectivity in CYP152 enzymes remains a challenge.

Purpose of the Study:

• To investigate the temperature-dependent catalytic profile and regioselectivity of the Nosocomiicoccus massiliensis peroxygenase (OleT_NS).
• To elucidate the structural and dynamic mechanisms underlying OleT_NS's selective fatty acid decarboxylation.
• To establish a new model for P450 enzyme reactivity and guide the engineering of selective biocatalysts.

Main Methods:

• Characterization of OleT_NS enzyme activity across a range of temperatures.
• Structural and dynamic analyses, including investigation of loop motions and hydrogen-bonding networks.
• Comparative analysis with other CYP152 family members and cold-active enzymes.

Main Results:

• OleT_NS exhibits unique temperature-dependent regioselectivity, with enhanced β-regioselectivity at milder temperatures.
• Coordinated motions between the F-G loop and His85 promote substrate burial and favor decarboxylation at lower temperatures.
• Elevated temperatures disrupt these coordinated motions, leading to altered hydrogen bonding and reduced alkene yields.
• The enzyme's flexibility and charged surface resemble cold-active enzymes, contributing to catalytic control.

Conclusions:

• OleT_NS demonstrates a temperature-dependent switch mechanism governing its catalytic selectivity.
• Structural dynamics and thermodynamic adaptation play critical roles in shaping P450 enzyme reactivity.
• This study provides principles for engineering selective biocatalysts for sustainable alkene production.