Abstract
The cytochrome P450 superfamily are heme-dependent monooxygenases that catalyze the chemically challenging activation of molecular oxygen following a common pathway with the formation of highly reactive iron(IV) oxo species. Cytochrome P450cam (CYP101A1), from the soil-bacterium Pseudomonas putida is one of the best characterized cytochromes P450, catalyzes the hydroxylation of d-camphor to 5-exo-hydroxycamphor. Wild type (WT) P450cam has a well-defined substrate selectivity and product specificity, which makes it an excellent probe of the role of enzyme structure in determining regioselectivity. This work focuses on multisite P450cam mutants at active site residues designed with the goal to promote changes in substrate orientation and/or location that might give rise to different hydroxylation products. Mutants were designed to maintain the same atom balance as WT, so as not to change the active site volume. In vitro site-directed mutations on CYP101A1 were done to examine enzyme activity and substrate turnover product. Structural perturbations in camphor-bound mutants were probed using 1H-15N TROSY-HSQC. Results show all mutant enzymes preserved the regio- and stereospecificity of camphor hydroxylation at 5-exo position with varying degrees of efficiency, suggesting the enzyme core residues are not the sole contributors to P450cam conservation of hydroxylation product.