Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents.
Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.
Wong
Tuck Seng
TS
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, California 91125, USA.
Arnold
Frances H
FH
Schwaneberg
Ulrich
U
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D019020:Directed Molecular Evolution / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005663:Furans / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase; D009930:Organic Chemicals / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D012996:Solutions; D012997:Solvents / Q000737:chemistry*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D005663:Furans; D009930:Organic Chemicals; D012996:Solutions; D012997:Solvents; C018674:tetrahydrofuran; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D004121:Dimethyl Sulfoxide