Guided by postulations in the relationship between structure and functionality

a saturation mutagenesis was performed on the D168 position of a P450 BM-3 (A74G/F87V/L188Q/E435T) mutant which originally transformed indole largely into indigo. Two novel mutants have been obtained based on the differences in color and absorption spectrum of the indole-hydroxylation products followed by product composition analysis using HPLC. One mutant

P450 BM-3 (A74G/F87V/L188Q/E435T/D168W) catalyzes indole into indirubin in 90% with a decrease of kcat/Km in 80%

mainly owing to its Km is increased by 4.8 times as compared with the parent. The other one

P450 BM-3 (A74G/F87V/L188Q/E435T/D168R) transforms indole into indigo in about 87%

which is higher than the parent and its kcat/Km has an increase in over 1.37 times as that of the parent. Such results indicate that the amino acid residue substitution on the D168 position combined with the mutation of E435T can influence the hydroxylation regioselectivity of P450 BM-3 as well as catalytic activity

which determines indole-hydroxylation product as indigo or indirubin. Such influence could not be caused by the mutation on a single amino acid residue site

neither E435 nor D168

because of the lack in some synergistic effect. In conclusion

the directed evolution guided by rational hypothesis for engineering the catalytic properties of P450 BM-3 has succeeded in getting two mutants exhibiting novel characteristics for indole-hydroxylation.