Mechanism of Sugar Ring Contraction and Closure Catalyzed by UDP-D-apiose/UDP-D-xylose Synthase (UAXS)

Uridine diphosphate (UDP)-apiose/UDP-xylose synthase (UAXS) is a member of the short-chain dehydrogen-ase/reductase superfamily (SDR), which catalyzes the ring contraction and closure of UDP-D-glucuronic acid (UDP-GlcA), affording UDP-apiose and UDP-xylose. UAXS is a special enzyme that integrates ring-opening, decarboxylation, rearrangement, and ring closure/contraction in a single active site. Recently, the ternary complex structure of UAXS was crystallized from Arabidopsis thaliana. In this work, to gain insights into the detailed formation mechanism of UDP-apiose and UDP-xylose, an enzyme-substrate reactant model has been constructed and quantum mechanical/ molecular mechanical (QM/MM) calculations have been performed. Our calculation results reveal that the reaction starts from the C4-OH oxidation, which is accompanied by the conformational transformation of the sugar ring from chair type to boat type. The sugar ring-opening is prior to decarboxylation, and the deprotonation of the C2-OH group is the prerequisite for sugar ring-opening. Moreover, the keto-enol tautomerization of the decarboxylated intermediate is a necessary step for ring closure/contraction. Based on our calculation results, more UDP-apiose product was expected, which is in line with the experimental observation. Three titratable residues, Tyr185, Cys100, and Cys140, steer the reaction by proton transfer from or to UDP-GlcA, and Arg182, Glu141, and D337 constitute a proton conduit for sugar C2-OH deprotonation. Although Thr139 and Tyr105 are not directly involved in the enzymatic reaction, they are responsible for promoting the catalysis by forming hydrogen-bonding interactions with GlcA. Our calculations may provide useful information for understanding the catalysis of the SDR family.