Structural and Mechanistic Insights into 4′-Dehydrogenation Catalyzed by AprD5 in Apramycin Biosynthesis

Abstract

AprD5 is a member of the NAD⁺-dependent short-chain dehydrogenase/reductase (SDR) superfamily. It participates in the apramycin biosynthetic pathway by catalyzing 4′-oxidation of UDP-β-d-glucose (UBG) to initiate the assembly of UDP-4′-amino-4′-deoxy-β-d-glucose in partnership with the aminotransferase AprL. Here, we report a 1.9 Å crystal structure of AprD5 in complex with NAD⁺ and its substrate UBG, which reveals a well-ordered ternary complex poised for catalysis. The cofactor and substrate are precisely aligned for hydride transfer by a conserved Ser–Tyr–Lys catalytic triad, while the nucleotide-sugar is constrained in a single reactive conformation through an extensive hydrogen-bonding network and a tight electrostatic clamp on the diphosphate group. Although 4′-oxidation is a common initiating step in SDR-catalyzed 4′-epimerization and 4′,6′-dehydration, the structural features that direct these closely related enzymes toward distinct reaction outcomes remain poorly understood. Comparative structural analysis reveals that AprD5 lacks the catalytic architecture required for dehydration while a rigid phosphate clamp and extensive substrate-anchoring network restrict the conformational rearrangements necessary for epimerization. Together, these features suggest how a conserved SDR scaffold is specialized for dedicated 4′-dehydrogenation and highlight general structural principles that may govern catalytic divergence among nucleotidyl-sugar SDR enzymes.