Abstract
Cytochromes P450 are a superfamily of heme-containing monooxygenases involved in a variety of oxidative metabolic reactions, primarily catalyzing the insertion of an oxygen atom into a C-H bond. CYP102 represents the first example of a bacterial P450 that can be classified as a type II (eukaryotic-like) P450 and functions as a catalytically self-sufficient enzyme. These unique features have made CYP102 an attractive system for studying P450 structure and function. However, an overall picture of the specific amino acid residues that are crucial to the functioning of CYP102 and the effect of mutations on the P450 structure and catalysis is yet to be reported. Such an approach will aid protein engineering approaches used to improve this enzyme. To address this research knowledge gap, we have investigated 105 CYP102 crystal structures in this study. We demonstrate that the CYP102 active site is highly dynamic and flexible. Amino acid residues that play critical roles in substrate binding, orientation, and anchoring were identified. Mutational studies highlighted the roles of amino acids and provided possible bioengineering improvement strategies for CYP102. Decoy molecules are a promising agent for deceiving CYP102 and permitting non-native substrates into the active site. Ru(II)-diimine photosensitizers and zinc/cobalt (III) sepulchrate (Co(III)Sep) could be used as alternative electron sources. The present study serves as a reference for understanding the structure-functional analysis of CYP102 family members precisely and of P450 enzymes in general. Significantly, this work contributes to the effort to develop an improved CYP102 enzyme, thereby advancing the field of P450 research and potentially leading to new industrial applications.