Fig. 1.

Test of UDP-xylose synthase and UDP-galactose epimerase activities. UDP-xylose synthase (UXS) and UDP-galactose/glucose epimerase (GalE1) activities were assayed by incubating the enzyme (1 μl purified enzyme or 5 μl E. coli crude extract) in the presence of the relevant 3 mM UDP-sugar and 3 mM NAD⁺ in 80 mM Tris–Cl, pH 7.7 at 30 or 37 °C (final volume 50 μL). (A) SAX-HPLC of 4 h assays performed using UDP-glucuronic acid as substrate and lysates of bacteria expressing T. vaginalis UXS, Drosophila GMD (negative control) or Cryptococcus UXS (positive control). After injection onto a Hypersil column (0.5 × 25 cm), the column was washed for 10 min with buffer A (2 mM ammonium formate, pH 3.2; the flow rate was 1.5 ml/min) prior to elution with a linear gradient from 0 to 40% buffer B (600 mM ammonium formate, pH 3.2) as described [7]; absorbance at 254 nm was recorded. Standard UDP-xylose elutes at around 6 min as compared to the UDP-glucuronic acid at 7 min (Stds). The peak at 4.5 min corresponds to imidazole present in the lysis buffer. (B) Ion-pair RP-HPLC of assays of recombinant UXS in either purified form or in an E. coli extract (+IPTG) showing conversion of UDP-GlcA to UDP-Xyl. A control extract of E. coli transformed with the UXS plasmid but not induced (-IPTG) showed no such activity. Analysis was performed using a Cosmosil C18-AR-II column (250 mm × 4.6 mm; Nacalai Tesque, Kyoto, Japan): buffer A was 20 mM triethylamine-acetate (pH 7) and buffer B was 20 mM triethylamine-acetate (pH 7) containing 10% acetonitrile [19]. After isocratic elution with 100% buffer A, a gradient of 1% per minute (buffer B) was applied after 15 min. (C) Negative-mode MALDI-TOF MS of pooled UXS products; the m/z of UDP-xylose [M-H]⁻ of 535.3 compares to the calculated molecular mass of 536.2. For analysis, 1 μl of an aliquot of UDP-Xyl was spotted onto a MALDI plate, vacuum dried prior to application of 1 μl 2,5-dihydroxybenzoic acid (DHB; 2% in 30% acetonitrile/70% 50 mM (NH₄)₂SO₄); the dried and crystallized sample was analysed by MALDI-TOF MS using a Bruker Ultraflex instrument. (D) Ion-pair RP-HPLC of GalE1 assays; purified recombinant T. vaginalis GalE converted UDP-Gal partly into UDP-Glc and also UDP-Glc partly into UDP-Gal, whereas purified recombinant T. vaginalis UXS displayed no such activity.