Structural Characterization of Glucurono-xylooligosaccharides

Substrates consisted of glucurono-xylans or glucurono-xylooligosaccharides. The glucurono-xylooligosaccharides UX and UXXX were purchased from Megazyme International (County Wicklow, Ireland), whereas UXX and XUXX were made as follows: 5 g of birch wood xylan (Sigma) was digested to completion with either CjXyn10A (19 (link)) or NpXyn11A (20 (link)) xylanases in 50 mm sodium phosphate, 12 mm citrate buffer, pH 6.5, containing 1 mg/ml of BSA (PC buffer) to completion. The glucurono-xylooligosaccharides generated by CjXyn10A, UXX, or NpXyn11A, XUXX, were purified from neutral xylooligosaccharides using Dowex chromatography (9 (link)). The structure of the products were confirmed by incubation with CjGlcA67A, which generated xylotriose from the CjXyn10A product but no undecorated xylooligosaccharide from the NpXyn11A product, whereas BoAgu115A released xylotriose and xylotetraose from glucurono-xylooligosaccharides generated by the GH10 and GH11 xylanases, respectively. The deduced structure of the two glucurono-xylooligosaccharides is entirely consistent with the glucuronoxylan binding mode of the two xylanases (21 (link), 22 (link)). Enzyme assays, in which polysaccharides or glucurono-xylooligosaccharides were the substrates, were carried out in PC buffer at 37 °C using enzyme purified to electrophoretic homogeneity by immobilized metal ion affinity chromatography. The concentration of enzyme varied from 10 nm for the wild type glucuronidase to 10 μm for the least active variants of BoAgu115A. For kinetic assays, glucuronic acid was detected using the α-d-glucuronidase assay kit in which the uronic acid released is oxidized to glucarate with the concomitant reduction of NAD⁺ to NADH, which was monitored continuously at 340 nm and quantified using a molar extinction coefficient of 6220 m⁻¹ cm⁻¹. The molar concentration of the GlcA/MeGlcA in the glucuronoxylans was quantified by digesting 100 μg of the polysaccharides to completion with BoAgu115A. To measure the activity of the BoAgu115A mutants the xylotetraose reaction product released from XUXX was monitored by high performance anion-exchange chromatography (HPAEC) as described previously (18 (link)). The reaction was carried out in 20 mm sodium phosphate buffer, pH 7.0, at a substrate concentration (1 mm) that was well below the K_M. Thus, the initial rate of hydrolysis of the glucurono-xylooligosaccharides gives a direct readout of k_cat/K_M (19 (link)). The glucuronidase-catalyzed reactions were also subjected to Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE)⁴ as follows: a alcohol-insoluble residue was prepared from mature Arabidopsis thaliana wild type and gux1gux2 stems as well as wild type willow, barley, sugar cane, and Miscanthus stems, as previously described (23 (link)). Alcohol-insoluble residue (500 μg) was pre-treated with 20 μl of 4 m NaOH for 1 h, neutralized with HCl, and ammonium acetate buffer, pH 6.0, added to a final concentration of 0.1 m and a final volume of 500 μl. The Arabidopsis alcohol-insoluble residue was digested to completion with xylanases (CjXyn10A and NpXyn11A), a glucuronoxylanase (BoGH30; Bacova_03432), and GH67 and GH115 α-glucuronidases (CjGlcA67A and BoAgu115A) as stated in the text and then dried in vacuum, whereas the other xylans were just digested with BoAgu115A. Released mono- and oligosaccharides were labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid (Invitrogen) and separated by gel electrophoresis as previously described (24 (link)). Xylooligosaccharides (Xyl_1–6, Megazyme Int.) were derivatized alongside each set of samples and run as standards within each gel.