Biotransformation of beta-ionone by engineered cytochrome P450 BM-3.
Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for beta-ionone (<1 min(-1)). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in beta-ionone hydroxylation activity (115 min(-1)). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-beta-ionone with an optical purity of 39%. All mutants regioselectively converted beta-ionone into 4-hydroxy-beta-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. itbvur@itb.uni-stuttgart.de
Vlada B
VB
Urlacher
Akhmadjan
A
Makhsumkhanov
Rolf D
RD
Schmid
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D045792:Norisoprenoids / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering
D045792:Norisoprenoids; D003577:Cytochrome P-450 Enzyme System; C008157:beta-ionone