Detalle de: Origins of stereoselectivity in evolved ketoreductases

Significance Ketoreductases are the most commonly used enzymes in industrial pharmaceutical synthesis. We investigated the nature of enantioselectivity in closely related mutant ketoreductases that reduce almost-symmetrical 3-oxacyclopentanone and 3-thiacyclopentanone, which are difficult to reduce enantioselectively by other means. We present the efficiencies of select variants and their crystallographic structures. Our experimental and theoretical studies reveal how mutations modulate the stereoselectivity of the reduction. Molecular dynamics simulations of the Michaelis–Menten and transition state-bound complexes were used to rationalize the observed stereochemical outcomes. We discovered that the closed conformation of the flexible substrate binding loop is likely the catalytically active one imparting the stereochemical preferences. Our molecular dynamics approach reveals how each enzyme stabilizes the diastereomeric transition structures by altering the active site size.