- Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst
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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, Leu 188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indiruhin at a catalytic efficiency of 1365M-1s-1 (kcat=2.73 s-1 and Km,=2.0mM). 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.
- Li, Qing-Shan,Schwaneberg, Ulrich,Fischer, Peter,Schmid, Rolf D.
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p. 1531 - 1536
(2007/10/03)
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