Abstract
The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-beta-l-threo-pentopyranosyl-4''-ulose in the presence of NAD(+). The second activity converts UDP-beta-l-threo-pentopyranosyl-4''-ulose and NADH to UDP-xylose and NAD(+), albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.
Highlights
Merly Pseudomonas aeruginosa) [10, 11]; in the gum-like polysaccharides secreted by Aeromonas nichidenii [12]; and recently in the O-antigenic polysaccharide of the Gram-negative plant endosymbiont Rhizobium leguminosarum [13]
We report the first identification of a gene in the plant pathogen Gram-negative bacterium Ralstonia solanacearum str
GMI1000, which encodes an enzyme capable of converting UDP-GlcA to UDP-4-keto-pentose and subsequently at lower rate to UDP-xylose (Fig. 1B). The identification of this enzyme provides new insights related to UDP-GlcA metabolism and may help to explain how early plants or other eukaryotes obtained UDP-GlcA decarboxylase (Uxs) or Uaxs genes
Summary
Phylogeny Analysis of Plant Uxs and Uaxs Homologs—The BLAST program [20] was used to identify proteins in the NCBI nonredundant data base that share high sequence similarity to functional plant Uaxs. HPLC-based Enzyme Assay—Unless otherwise indicated, RsU4kpxs reactions (final volume, 50 l) consisted of 50 mM sodium phosphate, pH 7.6, 1 mM NADϩ, 1 mM UDP-GlcA, and 1 g of recombinant RsU4kpxs. The D2O reactions were performed in a final mixture of D2O:H2O (9:1 v/v) and contained 50 mM sodium phosphate, pH, pD 7.6, 1 mM UDP-GlcA, 1 mM NADϩ, and 3 g of recombinant RsU4kpxs. Spectra of individual 1 mM standards of NADϩ, UDP-xylose, UDP-GlcA, and NADH prepared in the same buffer were collected with the same temperature and parameter settings. Inhibition assays were performed by first mixing the enzyme and sodium phosphate buffer with various additives (e.g. nucleotides) on ice for 10 min. The activities were terminated (100 °C), and the amount of UDP-4-keto-pentose was measured by HPLC
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