Probing of the reaction pathway of human UDP-xylose synthase with site-directed mutagenesis.
Uridine 5'-diphosphate (UDP)-xylose (UDP-Xyl) synthase (UXS) catalyzes the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcUA) to UDP-Xyl. The closely related UDP-glucuronic acid 4-epimerase (UGAE) interconverts UDP-GlcUA and UDP-galacturonic acid (UDP-GalUA) in a highly similar manner via the intermediate UDP-xylo-hexopyranos-4-uluronic acid (UDP-4-keto-GlcUA). Unlike UXS, however, UGAE prevents the decarboxylation. Human UXS (hUXS) and UGAE from Arabidopsis thaliana exhibit high structural similarity in the active site, but two catalytically important residues in hUXS (Glu(120) and Arg(277)) are replaced by Ser and Thr in the UGAE group. Additionally, Asn(176), which participates in substrate binding, is changed to Thr. We therefore analyzed single, double and triple mutants of hUXS carrying these substitutions to evaluate their significance for product specificity. All mutants showed considerably lower activities than wild-type hUXS (>1000-fold reduction). NMR spectroscopic analysis of the reaction products showed that UDP-β-L-threo-pentopyranos-4-ulose (UDP-4-keto-Xyl), UDP-Xyl or both, but no UDP-GalUA or UDP-4-keto-GlcUA were formed. Correlation of product characteristics, such as deuterium incorporation, with the amino acid replacements gave insights into structure-function relationships in UXS, suggesting that interaction between active site and overall enzyme structure rather than distinct conserved residues are decisive for product formation.
Institute of Biotechnology and Biochemical Engineering, NAWI Graz, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Thomas
T
Eixelsberger
Institute of Organic Chemistry, NAWI Graz, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria.
Hansjörg
H
Weber
Institute of Biotechnology and Biochemical Engineering, NAWI Graz, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria; Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria. Electronic address: bernd.nidetzky@tugraz.at.
Bernd
B
Nidetzky
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase
Decarboxylation; Epimerization; Sugar biosynthesis; UDP-galacturonic acid; UDP-xylose; UDP-xylose synthase