The asymmetric structure of human UDP-[alpha]-D-xylose synthase suggests a mechanism for regulating activity
Polizzi, Samuel Justin
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Proteoglycans provide structural integrity to connective tissues and facilitate growth factor binding to receptors. The first step in the formation of most proteoglycans is the covalent linkage of xylose to the hydroxyl of a serine on acceptor proteins. UDP-xylose (UDX) is the activated substrate required for xylose incorporation into the proteoglycan linker. UDX biosynthesis proceeds via the conversion of UDP-glucose to UDP-glucuronic acid (UGA) by UDP-glucose dehydrogenase (UGDH) and subsequent decarboxylation by UDP-xylose synthase (UXS). UDX is believed to regulate UDP-sugar pools by allosterically inhibiting UGDH and UXS. In order to understand how human UXS is regulated, we solved the 2.5A crystal structure of unliganded human UXS. UXS copurifies with the NAD+ cofactor tightly bound, unlike the only other structurally characterized UGA decarboxylase, E. coli ArnA. While ArnA is reported to bind NAD+ and UGA as substrates and release NADH and UDP-4-keto-xylose (UX4O) as products, UXS retains NADH and catalyzes a second hydride transfer to regenerate NAD+ and release UDX. We show that UXS will release the reaction intermediates UX4O and NADH in the presence of exogenous NAD+. The release of the reaction intermediates involves a cooperative conformational change that we show is conserved in ArnA. UXS activity is also stimulated >6-fold in the presence of a molecular crowding agent trimethylamine N-oxide. Solution studies show that UXS undergoes a concentration-dependent dimer-to-tetramer association (Kd = 4.68 μM), with the tetramer being the most active species. The crystal structure of UXS reveals a possible allosteric site that we predict would disrupt the tetramer. Combining these data suggests a model of regulation between high activity tetramers and low activity dimers mediated by a UDP-containing molecule.