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

P450 hydroxylation is widely used in steroidal drug synthesis, which can not only activate the oxidation of inert C-H bonds but also increase the pharmacological activity of steroidal compounds after hydroxylation modification. The two-step ordered selective hydroxylation of steroids catalyzed by P450 dihydroxylases often forms the key precursors or intermediates of steroid drugs. However, the poor catalytic performance of P450 dihydroxylase and the imbalance of two-step hydroxylation lead to the accumulation of intermediates, which restricts the efficient synthesis of dihydroxyl products. In this work, the hydroxylation sequence (first at C7 and then at C15) of DHEA to 7α,15α-diOH-DHEA catalyzed by CYP-cl3 from <i>Colletotrichum lini</i> 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.