Cytochrome P450s are the main enzymes responsible for the oxidative and reductive metabolism of numerous endogenous and xenobiotic substrates. The ability of the P450s to catalyze multiple types of reactions and their broad substrate specificity make these enzymes an excellent choice for engineering efforts to create new substrate specificities. We hope to engineer CYP119, the first P450 and only b-type hemeprotein to be identified thus far from any acidophilic, thermophilic archaebacteria, into a self-sufficient P450 capable of metabolizing non- natural substrates. CYP119 was originally cloned from Sulfolobus solfataricus, an aerobic, acidothermophilic archaeon, and identified as a potential P450 based n its sequence homology to other members of the P450 superfamily. Because of its acidophilic and thermophilic properties, CYP119 is a more versatile model system than those currently available. DOCK is an important tool that is helping to refine our understanding of P450 substrate specificity. DOCK has been tested with wild-type P450cam and two of its mutants. Further studies with a wider range of P450s and potential substrates are required to make the approach of practical utility. A homology model of CYP119 is being constructed based on alignments with the four crystallized P450s. Important residues in the active site will be identified using Midas. Site-directed mutagenesis will be used to assess their role in specificity and activity of the protein. We hope that the results from studies with P450cam, P450terp, P450eryf, P450BM-3 and CYP119 will provide the necessary information for the correct prediction of mutations that will effect desired changes in the catalytic specificity of P450s.
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