High Resolution NMR Studies of Cytochrome P450 Enzymes CYP101 and MycG

Cytochromes P450 are a remarkable superfamily of heme-thiolate monooxygenases capable of carrying out a chemical transformation that has been described as the "holy grail" of synthetic chemistry, the selective oxidation of inert C-H bonds. The desire to harness P450 oxidative chemistry for synthetic application drives enzyme engineering efforts from many scientists around the world. This work describes the sparsely implemented, yet powerful tool of solution NMR for identification of specific engineering targets using P450cam as model system. The technique holds value in its ability to implicate regions of a protein important for substrate binding that could not have been predicted by structural analysis, or any other method. Our work revealed that mutations of the NMR detected "hot spots" even remote from the active site result in altered substrate binding and enzyme activity for P450cam.;A cytochrome P450 enzyme dubbed MycG is involved in the biosynthesis of the macrolide antibiotic mycinamicin II, and is the focus of the remainder of this thesis. The project began with exploiting the paramagnetic relaxation enhancement of heme to orient the mycinamicin IV substrate molecule in the active site of MycG. This was done in order to refine the catalytically incompetent orientation captured by crystal structures. The model calculated from the NMR data illustrates that the substrate may adopt an orientation that differs from the crystal structure and corresponds better to the observed chemistry.;A detailed methodology for generating backbone resonance assignments for MycG is described. Over 75 % of MycG backbone resonance assignments were obtained allowing for extensive analysis of solution state structure and dynamics. Backbone N-H residual dipolar coupling measurements were obtained and applied as restraints for molecular dynamics simulations. Structural ensembles were generated which reflect experimental NMR data. The results affirm that global conformational changes take place depending on binding pocket occupancy, none of which are seen in the crystal structures.;A dynamic characterization of MycG was carried out by performing NMR monitored substrate titrations, hydrogen-deuterium exchange, and 15 N relaxation experiments. Regions sensitive to substrate binding were identified and their dynamics characterized. The general trend of MycG dynamics suggests that large scale, slow timescale movements couple with substrate binding both near and remote from the active site.