Abstract
Cytochromes P450 constitute a large superfamily of heme-thiolate proteins that catalyze a wide variety of reactions. Although they share similar structural folds, some P450s are highly substrate specific, while others are promiscuous. Cytochrome P450cam (CYP101A1) from Pseudomonas putida catalyzes the 5-exo-hydroxylation of camphor and requires the presence of redox partners: putidaredoxin (Pdx) and putidaredoxin reductase (PdR). Solution state nuclear magnetic resonance (NMR) experiments in the presence of non-native substrates revealed affected regions not only in the vicinity of the active site, as expected, but also in remote regions. From this we proposed a mechanism that incorporates these areas in CYP101A1’s conformational changes. In this study, we took the initial steps toward rationally re-engineering CYP101A1 to modify the substrate selectivity and product specificity. To achieve this, site directed mutagenesis was done to alter the protein structure. Heme spin state observation, enzymatic assays and gas chromatography were used to investigate the effects of the mutations, testing substrate binding and product turnover. A series of mutations were made simultaneously, but did not succeed. Despite the lack of success, insight was gained into the project’s future steps.