Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

UDP‐glucuronic acid (UDP‐GlcA) 4‐epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4‐oxidation of the substrate by NAD(+) and rotation of a decarboxylation‐prone β‐keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4‐keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4‐keto sugar has an undistorted (4) C (1) chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4‐keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP‐xylose synthases that decarboxylate UDP‐GlcA in stereospecific, configuration‐retaining reactions.