Structure and Mechanism of Human UDP-xylose Synthase: EVIDENCE FOR A PROMOTING ROLE OF SUGAR RING DISTORTION IN A THREE-STEP CATALYTIC CONVERSION OF UDP-GLUCURONIC ACID

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD⁺ and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-d-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the d-glucuronyl ring accommodated by UXS features a marked ⁴C₁chair to B_O,3boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C₄ (step 1) by aligning the enzymatic base Tyr¹⁴⁷ with the reactive substrate hydroxyl and it brings the carboxylate group at C₅ into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-d-glucuronic acid in the second chemical step. The protonated side chain of Tyr¹⁴⁷ stabilizes the enolate of decarboxylated C₄ keto species (²H₁half-chair) that is then protonated from the Si face at C₅, involving water coordinated by Glu¹²⁰. Arg²⁷⁷, which is positioned by a salt-link interaction with Glu¹²⁰, closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C₄ keto group by NADH, assisted by Tyr¹⁴⁷ as catalytic proton donor, yields UDP-xylose adopting the relaxed ⁴C₁chair conformation (step 3).