Analysis of the DXD Motifs in Human Xylosyltransferase I Required for Enzyme Activity

• Published in: The Journal of biological chemistry Vol. 279; no. 41; pp. 42566 - 42573
• Main Authors: Götting, Christian, Müller, Sandra, Schöttler, Manuela, Schön, Sylvia, Prante, Christian, Brinkmann, Thomas, Kuhn, Joachim, Kleesiek, Knut
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 08.10.2004
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Aspartic Acid - chemistry; Binding, Competitive; Blotting, Western; Cell Line; Cloning, Molecular; DNA Primers - chemistry; DNA, Complementary - metabolism; Glycine - chemistry; Glycosaminoglycans - chemistry; Heparin - chemistry; Humans; Insecta; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Pentosyltransferases - chemistry; Pentosyltransferases - physiology; Plasmids - metabolism; Polymerase Chain Reaction; Protamines - chemistry; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Sequence Homology, Amino Acid; Substrate Specificity; UDP Xylose-Protein Xylosyltransferase; Uridine Diphosphate - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M401340200

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the D X D motifs at positions 314–316 and 745–747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314 DED 316 motif did not have any effect on enzyme activity, whereas alterations of the 745 DWD 747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp 745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G ( K m = 6.9 μ m ) and D747E ( K m = 4.4 μ m ) in comparison with the wild-type enzyme ( K m = 0.9 μ m ). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower V max , with K m values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. K i values for inhibition by UDP were determined to be 1.9–24.6 μ m for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the K i constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.