Characterization Of The Reductive Properties Of Cytochrome P450 BM3

Cytochrome P450 BM3 is a well studied fatty acid monooxygenase. Unlike P450-enzymes in higher organisms, BM3 exhibits a natural peptide fusion of the P450 and the reductase domains. BM3 has shown an atypical reaction by reducing an aldehyde to its respective primary alcohol. Interestingly, initial results indicate that this previously unknown reduction can be enhanced by using a genetic variant of BM3, F87A T268A. BM3 wild type, the variant protein and the corresponding BM3 reductase domain were expressed in Escherichia coli and purified by affinity chromatography, characterized by cytochrome c reductase activity assays, heme and FAD/FMN content measurement (fluorescence, UV-Vis spectroscopy), purity (SDS-PAGE) and active heme (CO-binding) of each protein. Kinetic parameters were measured for the reduction of an aldehyde probe (2-Naphthaldehyde) using gas chromatography coupled with mass spectrometry. To investigate the mechanism of aldehyde reduction, the reaction was carried out under saturating CO conditions, in D2O buffers and under argon. This exciting new reductive property of P450 BM3 can be an important catalyst in biotechnology.