Modification Strategy of Two-Step Ordered Selective Hydroxylation of DHEA Catalyzed by CYP-cl3

ST-1 was revealed with the aid of computational analysis. To obtain highly C7α- and C15α-selective enzymes, we designed a three-step modification strategy that includes the high-throughput screening of C7-hydroxylation mutants, semirational design of C15-hydroxylation selectivity, and the combination of dominant mutation sites. As a result, we successfully obtained a dominant quadruple mutant A83P/E264I/V281A/T315P, whose the proportion of 7α,15α-diOH-DHEA reached 99.9%. The mechanism of key amino acid residues in improving the catalytic performance of CYP-cl3 was revealed by molecular docking and molecular dynamics simulation analysis. Our study guides the performance improvement of other P450 hydroxylase.