Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains.
Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase from Bacillus megaterium, is a multidomain protein containing heme, FAD, and FMN. Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme iron. The present study of the reconstitution of the monooxygenase activity of P450BM-3 utilized combinations of the different functional domains of P450BM-3. For this purpose, the FAD/NADPH- and FMN-binding domains of P450BM-3 as well as the combination of the heme- and FMN-binding domains (BMP/FMN) have been expressed and purified. The reconstitution systems, consisting of either BMP/FMN and FAD domains or BMP, FMN, and FAD domains, were still less effective than the holoenzyme, P450BM-3, but were much more effective than a system consisting of BMP and BMR. The maximal rate of oxidation of palmitic acid by the newly developed reconstitution systems is still only approximately 5% of the activity of the holoenzyme. The reconstitution systems produced omega-1, omega-2, and omega-3 monohydroxy palmitic acid, but not the secondary products of palmitic acid hydroxylation observed with the holoenzyme. The physical cause of the inability to reconstitute fully the maximal activity of the holoenzyme as well as the lack of secondary product formation is not presently understood.
Sevrioukova
I
I
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038, USA.
Truan
G
G
Peterson
J A
JA
D001410:Bacillus megaterium / Q000378:metabolism*; D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005486:Flavin Mononucleotide / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009251:NADPH-Ferrihemoprotein Reductase; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019308:Palmitic Acid; D006418:Heme; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases