Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity.
Cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of substituted and unsubstituted aromatics. However, since activities were quite low, a combination of site-specific mutagenesis and directed evolution was applied to produce 7800 variants of CYP116B3, which were screened via a newly developed high-throughput screening system based on the dealkylation of 7-ethoxycoumarin catalyzed by recombinant E. coli. The best mutant was found after four rounds of directed evolution and had a 240-fold increased deethylation activity toward 7-ethoxycoumarin (223 nmol product/nmol P450.min) and a 10-fold increased demethylation activity toward 7-methoxycoumarin (9 nmol product/nmol P450.min).
Liu
Luo
L
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Schmid
Rolf D
RD
Urlacher
Vlada B
VB
D003374:Coumarins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D003640:Dealkylation; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology*
D003374:Coumarins; D011994:Recombinant Proteins; C017299:7-ethoxycoumarin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases