Peroxidase activity of bacterial cytochrome P450 enzymes: modulation by fatty acids and organic solvents.
The modulation of peroxidase activity by fatty acid additives and organic cosolvents was determined and compared for four bacterial cytochrome P450 enzymes, thermostable P450 CYP119A1, the P450 domain of CYP102A1 (BMP), CYP152A1 (P450(bsbeta)), and CYP101A1 (P450(cam)). Utilizing a high-throughput microplate assay, we were able to readily screen more than 100 combinations of enzymes, additives and cosolvents in a convenient and highly reproducible assay format. We found that, in general, CYP119A1 and BMP showed an increase in peroxidative activity in the presence of fatty acids, whereas CYP152A1 revealed a decrease in activity and CYP101A1 was only slightly affected. In particular, we observed that the conversion of the fluorogenic peroxidase substrate Amplex Red by CYP119A1 and BMP was increased by a factor of 38 or 11, respectively, when isopropanol and lauric acid were present in the reaction mixture. The activity of CYP119A1 could thus be modulated to reach more than 90% of the activity of CYP152A1 without effectors, which is the system with the highest peroxidative activity. For all P450s investigated we found distinctive reactivity patterns, which suggest similarities in the binding site of CYP119A1 and BMP in contrast with the other two proteins studied. Therefore, this study points towards a role of fatty acids as activators for CYP enzymes in addition to being mere substrates. In general, our detailed description of fatty acid- and organic solvent-effects is of practical interest because it illustrates that optimization of modulators and cosolvents can lead to significantly increased yields in biocatalysis.
Rabe
Kersten S
KS
Universität Dortmund, Fachbereich Chemie, Biologisch-chemische Mikrostrukturtechnik, Dortmund, Germany.
Erkelenz
Michael
M
Kiko
Kathrin
K
Niemeyer
Christof M
CM
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D015227:Lipid Peroxidation; D009930:Organic Chemicals / Q000737:chemistry; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry
D001426:Bacterial Proteins; D005227:Fatty Acids; D009930:Organic Chemicals; D010078:Oxazines; D012997:Solvents; C470430:Amplex Red; D003577:Cytochrome P-450 Enzyme System; D009195:Peroxidase