Directed evolution of the fatty-acid hydyoxylase P450BM-3 into an indole-hydroxylating catalyst

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium ca talyzes subterminal hydroxylation of saturated long-chain fatty acids and s tructurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent mo del 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 t o hydroxylate indole, producing indigo and indirubin at a catalytic efficie ncy of 1365 m(-1) s(-1) (k(cat) = 2.73 s(-1) and K-m=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 en zyme can be engineered to catalyze the transformation of substrates with st ructures widely divergent from those of its native substrate.