Dataset Information

Human UDP-?-D-xylose synthase and Escherichia coli ArnA conserve a conformational shunt that controls whether xylose or 4-keto-xylose is produced.

ABSTRACT: Human UDP-?-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-?-D-glucuronic acid (UGA), and then reducing the UDP-?-D-4-keto-xylose (UX4O) to produce UDP-?-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.

SUBMITTER: Polizzi SJ

PROVIDER: S-EPMC4932848 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

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.

Polizzi Samuel J SJ Walsh Richard M RM Peeples William B WB Lim Jae-Min JM Wells Lance L Wood Zachary A ZA

Biochemistry 20121029 44

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-keto-xylose (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 fr ...[more]

PMID: 23072385

Similar Datasets

Project description:UDP-xylose synthase (Uxs1) is strongly conserved from bacteria to humans, but because no mutation has been studied in any animal, we do not understand its roles in development. Furthermore, no crystal structure has been published. Uxs1 synthesizes UDP-xylose, which initiates glycosaminoglycan attachment to a protein core during proteoglycan formation. Crystal structure and biochemical analyses revealed that an R233H substitution mutation in zebrafish uxs1 alters an arginine buried in the dimer interface, thereby destabilizing and, as enzyme assays show, inactivating the enzyme. Homozygous uxs1 mutants lack Alcian blue-positive, proteoglycan-rich extracellular matrix in cartilages of the neurocranium, pharyngeal arches, and pectoral girdle. Transcripts for uxs1 localize to skeletal domains at hatching. GFP-labeled neural crest cells revealed defective organization and morphogenesis of chondrocytes, perichondrium, and bone in uxs1 mutants. Proteoglycans were dramatically reduced and defectively localized in uxs1 mutants. Although col2a1a transcripts over-accumulated in uxs1 mutants, diminished quantities of Col2a1 protein suggested a role for proteoglycans in collagen secretion or localization. Expression of col10a1, indian hedgehog, and patched was disrupted in mutants, reflecting improper chondrocyte/perichondrium signaling. Up-regulation of sox9a, sox9b, and runx2b in mutants suggested a molecular mechanism consistent with a role for proteoglycans in regulating skeletal cell fate. Together, our data reveal time-dependent changes to gene expression in uxs1 mutants that support a signaling role for proteoglycans during at least two distinct phases of skeletal development. These investigations are the first to examine the effect of mutation on the structure and function of Uxs1 protein in any vertebrate embryos, and reveal that Uxs1 activity is essential for the production and organization of skeletal extracellular matrix, with consequent effects on cartilage, perichondral, and bone morphogenesis.

Project description:We recently reported that aldo-keto reductase 1B3-produced prostaglandin (PG) F(2?) suppressed the early phase of adipogenesis. PGE(2) is also known to suppress adipogenesis. In this study, we found that microsomal PGE(2) synthase (PGES)-1 (mPGES-1; PTGES1) acted as the PGES in adipocytes and that PGE(2) and PGF(2?) synergistically suppressed the early phase of adipogenesis. PGE(2) production was detected in preadipocytes and transiently enhanced at 3 h after the initiation of adipogenesis of mouse adipocytic 3T3-L1 cells, followed by a quick decrease; and its production profile was similar to the expression of the cyclooxygenase-2 (PTGS2) gene. When 3T3-L1 cells were transfected with siRNAs for any one of the three major PTGESs, i.e., PTGES1, PTGES2 (mPGES-2), and PTGES3 (cytosolic PGES), only PTGES1 siRNA suppressed PGE(2) production and enhanced the expression of adipogenic genes. AE1-329, a PTGER4 (EP4) receptor agonist, increased the expression of the Ptgs2 gene with a peak at 1 h after the initiation of adipogenesis. PGE(2)-mediated enhancement of the PTGS2 expression was suppressed by the co-treatment with L-161982, a PTGER4 receptor antagonist. Moreover, AE1-329 enhanced the expression of the Ptgs2 gene by binding of the cyclic AMP response element (CRE)-binding protein to the CRE of the Ptgs2 promoter; and its binding was suppressed by co-treatment with L-161982, which was demonstrated by promoter luciferase and chromatin immunoprecipitation assays. Furthermore, when 3T3-L1 cells were caused to differentiate into adipocytes in medium containing both PGE(2) and PGF(2?), the expression of the adipogenic genes and the intracellular triglyceride level were decreased to a greater extent than in medium containing either of them, revealing that PGE(2) and PGF(2?) independently suppressed adipogenesis. These results indicate that PGE(2) was synthesized by PTGES1 in adipocytes and synergistically suppressed the early phase of adipogenesis of 3T3-L1 cells in cooperation with PGF(2?) through receptor-mediated activation of PTGS2 expression.

Dataset Information

Human UDP-?-D-xylose synthase and Escherichia coli ArnA conserve a conformational shunt that controls whether xylose or 4-keto-xylose is produced.

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.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets