Extensive modelling and quantum chemical study of sterol C-22 desaturase mechanism: A commercially important cytochrome P450 family

Abstract

CYP450s, first wave biocatalysts and nature’s tool used for catalysing complex reactions like C-H bond hydroxylation, N-dealkylation, N-hydroxylation, O-dealkylation, etc. Engineered CYP450s catalyse new-to-nature carbene and nitrene transfer some of which have no precedent using chemical methods. Due to broad substrate range and immense biocatalytic scope, CYP450s are extensively used in synthesising of active pharmaceutical ingredients (APIs) and drugs. CYP450 desaturase adds double bonds to form mono and poly desaturated sterols. The addition of a double bond at a specific position is conserved within a particular structural fold type of CYP450. Minimal information is currently available on the mechanism of enzyme action of CYP450 desaturase, especially no information is available concerning experimental details on C-22 desaturase. Here, for the first time, we report the structure-function of desaturase and the detailed mechanism of the enzyme using quantum chemical (QC) calculations. The study provides atomistic details from precise near attack conformations of different sterols in the active site of CYP450 desaturase to the active site residues that define substrate specificity. A novel finding on second hydrogen abstraction; QC studies revealed transition states involving hydrogen abstractions. The second hydrogen abstraction takes place in a relay mode moving from carbon to carbon and then to electronegative oxygen attached to haem. Sterol diffusion path was predicted, and its plasticity was studied using extended molecular dynamic simulation experiments. This report is also a source of information for N-terminal modification and its effect on heterologous expression and the role of reductase binding for electron transfer. The results presented here will be valuable for guiding the experimental work to further improve the biocatalytic applicability of CYP450 desaturase enzymes.