title: Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.
abstract: Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via1H and13C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala79. In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site.
authors.affiliation: University of Vienna, Institute of Organic Chemistry, Währingerstrasse 38, A-1090 Vienna, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Puchner
authors.affiliation: Graz University of Technology, Institute of Biotechnology and Biochemical Engineering, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eixelsberger
authors.affiliation: Graz University of Technology, Institute of Biotechnology and Biochemical Engineering, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
authors.affiliation: University of Vienna, Institute of Organic Chemistry, Währingerstrasse 38, A-1090 Vienna, Austria. Electronic address: lothar.brecker@univie.ac.at.
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
mesh_terms: D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D060326:Chemistry Techniques, Synthetic; D006027:Glycosides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D012996:Solutions; D014574:Uronic Acids / Q000737:chemistry
chemical_list: D006027:Glycosides; D012996:Solutions; D014574:Uronic Acids; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase
keywords: 4-Keto-saccharides; Binding pattern; Human UDP-xylose synthase; Saturation transfer difference NMR