Human UDP-α-d-xylose Synthase and Escherichia coli ArnA Conserve a Conformational Shunt That Controls Whether Xylose or 4-Keto-Xylose Is Produced

Abstract

Human UDP-α-d-xylose synthase (hUXS) is a member of the short-chain dehydrogenase/reductase family of nucleotide-sugar modifying enzymes. hUXS contains a bound NAD+ cofactor that it recycles by first oxidizing UDP-α-d-glucuronic acid (UGA), and then reducing the UDP-α-d-4-ketoxylose (UX4O) to produce UDP-α-d-xylose (UDX). Despite the observation that purified hUXS contains a bound cofactor, it has been reported that exogenous NAD+ will stimulate enzyme activity. Here we show that a small fraction of hUXS releases the NADH and UX4O intermediates as products during turnover. The resulting apoenzyme can be rescued by exogenous NAD+, explaining the apparent stimulatory effect of added cofactor. The slow release of NADH and UX4O as side products by hUXS is reminiscent of the Escherichia coli UGA decarboxylase (ArnA), a related enzyme that produces NADH and UX4O as products. We report that ArnA can rebind NADH and UX4O to slowly make UDX. This means that both enzymes share the same catalytic machinery, but differ in the preferred final product. We present a bifurcated rate equation that explains how the substrate is shunted to the distinct final products. Using a new crystal structure of hUXS, we identify the structural elements of the shunt and propose that the local unfolding of the active site directs reactants toward the preferred products. Finally, we present evidence that the release of NADH and UX4O involves a cooperative conformational change that is conserved in both enzymes.

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution

“…X-ray analyses of UXS and ArnA identified domains (supplemental Fig. S1) involved in catalysis and cofactor binding (41,42). Protein sequence alignment (supplemental Fig.…”

Man o’ War Mutation in UDP-α-d-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

“…E 1 (residues 163−170) includes the short α-helix 165−168 , which has previously been shown to undergo a disorder-to-order transition upon substrate binding that shields the active site from solvent ( Figure 4A). 12 Given the intrinsic flexibility of helix 165−168 , it is possible that the disorder we observe in E 1 is not a direct result of the R236H and R236A substitutions. Because E 1 packs against E 2 , it is not unreasonable to assume that the unfolding of the latter element contributes to the greater flexibility of E 1 .…”

“…Here the mechanism is split into the productive and abortive pathways. 12 In the productive pathway (top) the UX4O is reduced to UDP-Xyl (UDX) and the NAD + cofactor is regenerated. In the abortive pathway (bottom) the E:NADH:UX4O complex releases the intermediates and the apoenzyme, E*, can be rescued by exogenous NAD + .…”

“…9−11 The recombinant enzyme lacking the transmembrane helix purifies with stoichiometric amounts of tightly bound NAD + cofactor that is recycled through successive oxidation and reduction steps in the catalytic mechanism ( Figure 1B). 12 In the first step, NAD + oxidizes UDP-α-D-glucuronic acid (UDPGlcA) to produce NADH and UDP-α-D-4-keto-xylose (UDP-4-keto-Xyl). Next, NADH reduces the ketose intermediate to produce UDP-Xyl.…”

“…During turnover the NADH and UDP-4-keto-Xyl intermediates can be released in an abortive cycle that generates the inactive apoenzyme ( Figure 1B). 12 The inactive enzyme can be rescued by adding exogenous NAD + to the reaction. 12 Because of the bifurcated mechanism, hUXS in the presence of saturating NAD + will produce both UDP-Xyl and the reaction intermediates NADH and UDP-4-keto-Xyl.…”