Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium-and long-chain fatty acids at the positions ω-1, ω- 2, and ω-3. A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to ω-oxycarboxylic acids and the chromophore p-nitrophenolate was developed. In contrast to the commonly used activity assays for this enzyme, relying on the consumption of oxygen or NADPH or the use of 14C- labeled carboxylic acids, the pNCA assay can even be used with crude extracts of the recombinant enzyme from lysed Escherichia coli cells. The kinetics of p-nitrophenolate formation are directly measured at a wavelength of 410 nm using a spectrophotometer or microtiter plate reader. Sensitivity of the assay is greatly enhanced if p-nitrophenoxydodecanoic or p- nitrophenoxypentadecanoic acid are used with the F87A mutant instead of the wild-type P450 BM-3 enzyme.
Bibliographical noteFunding Information:
1This work was supported by the BMBF (ZSP B3.3U), Bonn, Germany and by BASF AG, Ludwigshafen, Germany. 2 Current address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125. 3Abbreviations used: pNCA, p-nitrophenoxycarboxylic acid; DMSO, dimethyl sulfoxide; P450 BM-3, cytochrome P450 CYP102