title: Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.
abstract: The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.
authors.affiliation: Institut für Technische Biochemie, Universität Stuttgart, Germany. itbrsc@po.uni-stuttgart.de
authors.forename: Q S
authors.initials: QS
authors.lastname: Li
authors.forename: U
authors.initials: U
authors.lastname: Schwaneberg
authors.forename: P
authors.initials: P
authors.lastname: Fischer
authors.forename: R D
authors.initials: RD
authors.lastname: Schmid
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006900:Hydroxylation; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D007211:Indoles; C030374:indole; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; C027185:indirubin