Revisiting Carbonyl Reductase Stereocontrol: A Third Pocket Accommodates the E Ring in Podophyllotoxone‐Type Ketone Reduction

ABSTRACT Biocatalytic asymmetric reduction of ketones is one of the most cost‐effective, environmentally friendly, and stereoselective approaches for preparing chiral alcohols. However, the stereoselective reduction of bulky–bulky ketones remains a substantial challenge. In this study, we report the discovery and engineering of Sx PR 6980 , a carbonyl reductase (CR) from Streptomyces xanthophaeus GS‐BS18, for the stereoselective reduction of sterically demanding podophyllotoxone‐type substrates featuring a T‐shaped pentacyclic scaffold in which the quasi‐axial E ring imposes an additional steric constraint beyond the two groups flanking the carbonyl. Through Structure‐guided design, we identified two residue pairs—T149‐F153 and Q158‐M198—as key stereoselectivity switches. Crystal structures and molecular dynamics simulations revealed that the E ring occupies a functionally distinct third pocket. Remodeling this pocket, together with the region accommodating the fused A–D ring system, favored and stabilized a different near‐attack conformation, thereby altering the stereochemical outcome of hydride transfer from NADPH. This strategy was successfully extended to Sx PR 6980 isoenzymes with different intrinsic selectivities, demonstrating its applicability across both substrates and enzyme scaffolds. This work broadens the substrate scope of CRs toward bulky–bulky prochiral ketones and establishes a general paradigm for engineering CRs for complex polycyclic ketones.