Structure and Mechanism of Human UDP-xylose Synthase

Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, K.L.; Oppermann, U.; Brecker, Lothar; Nidetzky, Bernd

doi:10.1074/jbc.m112.386706

Cited by 36 publications

(69 citation statements)

References 51 publications

(54 reference statements)

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“…13 UXS is further classified as an ‘extended’ SDR due to an inserted nucleotide sugar binding domain (NSBD). 14,15 The extended SDR subfamily includes nucleotide sugar epimerases, dehydratases and decarboxylases, all of which use a similar NAD + -dependent mechanism. The recent crystal structure of human UXS (hUXS) in complex with NAD + and UDP has shed light on the mechanism (Figure 1).…”

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confidence: 99%

“…The recent crystal structure of human UXS (hUXS) in complex with NAD + and UDP has shed light on the mechanism (Figure 1). 15 Briefly, hUXS uses a bound NAD + cofactor to oxidize of the C4′ atom of UDP-α- d -glucuronic acid (UGA) to produce UDP-α- d -4-keto-glucuronic acid. The unstable β-keto-acid intermediate decarboxylates to form the more stable UDP-α- d -4-keto-xylose (UX4O).…”

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confidence: 99%

“…16 Despite this observation, several reports have demonstrated that adding exogenous NAD + can stimulate UXS activity by 10–104%. 15,17–21 This stimulation has been attributed to a significant contamination of apoenzyme in purified UXS. 15,17 …”

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confidence: 99%

“…15,17–21 This stimulation has been attributed to a significant contamination of apoenzyme in purified UXS. 15,17 …”

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confidence: 99%

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

Polizzi

Walsh²,

Peeples³

et al. 2012

Biochemistry

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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.

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confidence: 99%

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confidence: 99%

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confidence: 99%

“…15,17–21 This stimulation has been attributed to a significant contamination of apoenzyme in purified UXS. 15,17 …”

mentioning

confidence: 99%

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

Polizzi

Walsh²,

Peeples³

et al. 2012

Biochemistry

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“…Therefore, UXS has a crucial role for the conversion of diverse polysaccharides in the cell walls of plant, fungal, and bacterial glycans, and glycosaminoglycans in higher organisms (Reisset al, 1985;York and O'Neill, 2008;Coyne et al, 2011;Eixelsberger et al, 2012). At least three GhUXS genes are involved in the biosynthesis of fiber cell walls in cotton.…”

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confidence: 99%

Antisense expression of Gossypium hirsutum UDP-glucuronate decarboxylase in Arabidopsis leads to changes in cell wall components

et al. 2016

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ABSTRACT. UDP-glucuronate decarboxylase (UDP-xylose synthase; UXS, EC 4.1.1.35) is an essential enzyme of the non-cellulosic polysaccharide biosynthetic pathway. In the present study, using transient expression of fluorescently labeled Gossypium hirsutum UXS (GhUXS3) protein in onion epidermal cells, we observed that this protein was distributed in the cytoplasm. The GhUXS3 cDNA of cotton was expressed in an antisense orientation in Arabidopsis thaliana by Agrobacterium tumefaciensmediated transformation. Homozygous plants showing down-regulation of UXS were analyzed with northern blots. Compared to the untransformed control, transgenic plant showed shorter roots, earlier blossom formation, and delayed senescence. Biochemical analysis indicated that levels of rhamnose, mannose, galactose, glucose, xylose, and cellulose were reduced in some of the down-regulated antisense plants. These results suggest that GhUXS3 regulates the conversion of non-cellulosic polysaccharides and modulates their composition in plant cell walls. We also discuss a possible cellular function for GhUXS in determining the quality of cotton fibers.

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Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Eixelsberger

Nidetzky

2014

Adv Synth Catal

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Synthetic ways towards uridine 5′-diphosphate (UDP)-xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP-glucose 6-dehydrogenase (hUGDH) and UDP-xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP-α-xylose from UDP-glucose. In a mimic of the natural biosynthetic route, UDP-glucose is converted to UDP-glucuronic acid by hUGDH, followed by subsequent formation of UDP-xylose by hUXS. The nicotinamide adenine dinucleotide (NAD+) required in the hUGDH reaction is continuously regenerated in a three-step chemo-enzymatic cascade. In the first step, reduced NAD+ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10-phenanthrenequinone (PQ). Radical chemical re-oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo-enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP-xylose as well as an adequate oxygen supply for PQ re-oxidation as major bottlenecks of effective performance of the overall multi-step reaction system. Net oxidation of UDP-glucose to UDP-xylose by hydrogen peroxide (H2O2) could thus be achieved when using an in situ oxygen supply through periodic external feed of H2O2 during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g l−1) UDP-α-xylose. After two-step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi-step redox cascades in the synthesis of nucleotide sugar products.

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Structure and Mechanism of Human UDP-xylose Synthase

Cited by 36 publications

References 51 publications

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

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

Antisense expression of Gossypium hirsutum UDP-glucuronate decarboxylase in Arabidopsis leads to changes in cell wall components

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

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