Catalytic Promiscuity and Cascade Reaction Mechanism of Ketosteroid Isomerases for Addition Reactions of Cyclic Enones.

Steroidal medicines are a vital class of pharmaceuticals, with their active ingredients primarily derived from natural steroids. A key step in their biosynthesis involves the isomerization of ∆5-3-ketosteroids to ∆4-3-ketosteroids, catalyzed by ketosteroid isomerase (KSI). In this study, we discover that KSI exhibits an unexpected catalytic promiscuity, efficiently converting cyclic enones into homo- and hetero-addition products. Crystal structures of KSI variants and mutagenesis reveal the homo-dimeric interface as another active center. Molecular dynamics simulations and quantum mechanics/molecular mechanics calculations suggest a cascade catalytic mechanism by a single enzyme, in which isomerization occurs in the monomeric pocket, while the addition reaction preferentially occurs at the homo-dimeric interface due to reduced steric hindrance. These results not only provide valuable insights into the catalytic diversity of KSIs, but also inspire further exploration of new-to-nature and diverse catalytic properties of enzymes, thus expanding their applications in biocatalysis and drug development.