A Conformational Switch in the Catalytic Lysine Governs the Stereochemical Outcome of DERA-Catalyzed Aldol Addition

Abstract

2-Deoxyribose-5-Phosphate Aldolase (DERA) is a key enzyme in the pentose phosphate pathway. Due to its C-C bond formation and stereoselective capabilities, DERA has been widely used for biocatalytic applications including the synthesis of chiral intermediates for antiviral and anticancer drugs. Protein engineering has expanded its substrate-scope, improved yield, and enhanced S-stereoselectivity. Recently, rational mutagenesis of DERA from Geobacillus sp. (DERAGs) resulted in two enantiocomplementary variants, T12I/S185A and S185G. In particular, the single mutation at Ser185 to Gly inverts the natural S-selectivity to R-selectivity. However, the molecular basis of enantioselectivity remains unclear. Here, we determined high-resolution crystal structures of wild-type DERAGs and two enantiocomplementary variants in the apo form. Using a combination of structural biology, biochemistry, biocatalysis and molecular dynamic simulations, we show that the catalytic lysine adopts two conformations, a finding that relates to the enantioselectivity of DERA. We further identified a mechanism of regulating enantioselectivity via a key conserved amino acid. Using DERA from E. coli, we show that these findings are conserved across bacterial species.