Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose Synthase in the Plant Pathogenic Bacterium Ralstonia solanacearum Strain GMI1000, a Distinct Member of the 4,6-Dehydratase and Decarboxylase Family

Abstract

The UDP-sugar interconverting enzymes involved in UDPGlcA 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-␤-Lthreo-pentopyranosyl-4؆-ulose in the presence of NAD ؉ . The second activity converts UDP-␤-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.

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

“…1) was generated using the amino acid sequences of the UAS-like proteins (Fig. S1) together with other decarboxylases, including Arabidopsis UDP-xylose synthase (UXS), and two bacterial enzymes, a bifunctional UDP-4-keto-pentose/UDP-xylose synthase (RsU4kpxs) from the plant pathogen Ralstonia solanacearum and the C-terminal portion of ArnA that has a UDP-glucuronic acid 4-oxidase-6-decarboxylase activity (23,24). The UASs cluster into a group (III; see Fig.…”

“…The molecular mass of active recombinant SpUAS was estimated by size exclusion chromatography. Purified, recombinant SpUAS was eluted with dialysis buffer as eluent over a Superdex-75 100/300 GL column (23) that had been calibrated with proteins of known molecular mass (Bio-Rad).…”

“…Nucleotides and nucleotide sugars were detected by their A 261 nm (for UDP-sugars) and A 259 nm (for NAD ϩ ). The concentrations of reactants and products were determined by comparison of their peak areas with a calibration curve of standard UDP-GlcA (23).…”

UDP-Glucuronic Acid Decarboxylases of Bacteroides fragilis and Their Prevalence in Bacteria

“…This enzyme's N-terminal portion also has formyl transferase activity, and both activities participate in a pathway leading ultimately to the synthesis of UDP-4-amino-4-deoxy-L-arabinose (UDP-L-Ara4N). RsU4kpxs, from Ralstonia solanacearum (19), is similar to the C-terminal portion of ArnA, has UDP-GlcA decarboxylase activity, and produces UDP-xylose as an end product. However, much of the UDPxylose of this organism is further converted to UDP-L-Ara4N by a formyl transferase, similar to the N-terminal portion of ArnA, encoded by an adjacent gene.…”

“…Xylose is rarely described as a component of bacterial glycans. Bacterial molecules shown to contain xylose include the O antigen of Pseudomonas aeruginosa serotype O7 (10,25), the core oligosaccharide of the lipopolysaccharide (LPS) of Sinorhizobium meliloti (20), the LPS core oligosaccharide of Ralstonia solanacearum (19), the LPS of Leptospira species (23,35), and an S-layer glycoprotein of Parabacteroides distasonis (15).…”