CRYSTALLOGRAPHIC STUDIES ON CYTOCHROME P450

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The long range goal of this project is to study structure-function relationships in cytochromes P-450 and related proteins using x-ray crystallography, molecular biology, and biochemistry. The only known P-450 crystal structure is that of P-450cam. In addition, the structure of several substrate and inhibitor complexes plus one mutant structure are known. This information will be used to guide the design, production, and structure determination of site-directed variants for the purpose of probing the mechanism of P-450 action and the design of novel P-450 catalysts. What is known from these previous studies also will be used to design inhibitors of P-450cam in order to develop a model for the rational design of useful P-450 therapeutic agents. These will be synthesized and the binding constants and crystal structures of the P-450cam-inhibitor complexes determined. Work also will continue on new P-450s and related proteins. In particular is the P-45OBM-3 enzyme which hydroxylates fatty acids. This P-450 contains both the heme domain and the FAD/FMN P450 reductase domain in a single polypeptide chain. Sequence comparisons also indicate that this bacterial P-450 is a good model for eukaryotic P-450s. The crystal structure of the entire protein and its domains will be determined. Other crystallographic projects include a P-450 which demethylates aromatic methoxy acids (P-450RR1) and the linalool hydroxylase (P-450lin). The common theme of all these projects is to better understand what structural features control substrate specificity and inter-protein electron transfer reactions in P-450s. Results from these studies should also have practical relevance in the design of P-450 inhibitors as useful therapeutic agents and the design of novel hydroxylases.