A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A.
Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the positions omega-1, omega-2, and omega-3. A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-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.
Schwaneberg
U
U
Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, Stuttgart, 70569, Germany.
Schmidt-Dannert
C
C
Schmitt
J
J
Schmid
R D
RD
D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D020134:Catalytic Domain / Q000235:genetics; D003124:Colorimetry / Q000379:methods* / Q000706:statistics & numerical data; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000138:chemical synthesis / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods* / Q000706:statistics & numerical data; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases