Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity - PubMed (original) (raw)
. 2004 Oct 8;279(41):42566-73.
doi: 10.1074/jbc.M401340200. Epub 2004 Aug 4.
Affiliations
- PMID: 15294915
- DOI: 10.1074/jbc.M401340200
Free article
Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity
Christian Götting et al. J Biol Chem. 2004.
Free article
Abstract
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 DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 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 Asp745 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 (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km 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. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.
Similar articles
- Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.
Müller S, Disse J, Schöttler M, Schön S, Prante C, Brinkmann T, Kuhn J, Kleesiek K, Götting C. Müller S, et al. Biochem J. 2006 Feb 15;394(Pt 1):163-71. doi: 10.1042/BJ20051606. Biochem J. 2006. PMID: 16225459 Free PMC article. - Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity.
Müller S, Schöttler M, Schön S, Prante C, Brinkmann T, Kuhn J, Götting C, Kleesiek K. Müller S, et al. Biochem J. 2005 Mar 1;386(Pt 2):227-36. doi: 10.1042/BJ20041206. Biochem J. 2005. PMID: 15461586 Free PMC article. - Molecular cloning and expression of human UDP-d-Xylose:proteoglycan core protein beta-d-xylosyltransferase and its first isoform XT-II.
Götting C, Kuhn J, Zahn R, Brinkmann T, Kleesiek K. Götting C, et al. J Mol Biol. 2000 Dec 8;304(4):517-28. doi: 10.1006/jmbi.2000.4261. J Mol Biol. 2000. PMID: 11099377 - Human xylosyltransferases in health and disease.
Götting C, Kuhn J, Kleesiek K. Götting C, et al. Cell Mol Life Sci. 2007 Jun;64(12):1498-517. doi: 10.1007/s00018-007-7069-z. Cell Mol Life Sci. 2007. PMID: 17437056 Free PMC article. Review. - The never-ending story of peptide O-xylosyltransferase.
Wilson IB. Wilson IB. Cell Mol Life Sci. 2004 Apr;61(7-8):794-809. doi: 10.1007/s00018-003-3278-2. Cell Mol Life Sci. 2004. PMID: 15095004 Free PMC article. Review.
Cited by
- Recent advances on glycosyltransferases involved in the biosynthesis of the proteoglycan linkage region.
Gao J, Huang X. Gao J, et al. Adv Carbohydr Chem Biochem. 2021;80:95-119. doi: 10.1016/bs.accb.2021.10.003. Epub 2021 Nov 24. Adv Carbohydr Chem Biochem. 2021. PMID: 34872657 Free PMC article. Review. - Systems identification and characterization of β-glucuronosyltransferase genes involved in arabinogalactan-protein biosynthesis in plant genomes.
Ajayi OO, Showalter AM. Ajayi OO, et al. Sci Rep. 2020 Nov 25;10(1):20562. doi: 10.1038/s41598-020-72658-4. Sci Rep. 2020. PMID: 33239665 Free PMC article. - Biallelic variants in KYNU cause a multisystemic syndrome with hand hyperphalangism.
Ehmke N, Cusmano-Ozog K, Koenig R, Holtgrewe M, Nur B, Mihci E, Babcock H, Gonzaga-Jauregui C, Overton JD, Xiao J, Martinez AF, Muenke M, Balzer A, Jochim J, El Choubassi N, Fischer-Zirnsak B, Huber C, Kornak U, Elsea SH, Cormier-Daire V, Ferreira CR. Ehmke N, et al. Bone. 2020 Apr;133:115219. doi: 10.1016/j.bone.2019.115219. Epub 2020 Jan 7. Bone. 2020. PMID: 31923704 Free PMC article. - Enzymatic Activity of Xyloglucan Xylosyltransferase 5.
Culbertson AT, Chou YH, Smith AL, Young ZT, Tietze AA, Cottaz S, Fauré R, Zabotina OA. Culbertson AT, et al. Plant Physiol. 2016 Jul;171(3):1893-904. doi: 10.1104/pp.16.00361. Epub 2016 May 20. Plant Physiol. 2016. PMID: 27208276 Free PMC article. - XYLT1 mutations in Desbuquois dysplasia type 2.
Bui C, Huber C, Tuysuz B, Alanay Y, Bole-Feysot C, Leroy JG, Mortier G, Nitschke P, Munnich A, Cormier-Daire V. Bui C, et al. Am J Hum Genet. 2014 Mar 6;94(3):405-14. doi: 10.1016/j.ajhg.2014.01.020. Epub 2014 Feb 27. Am J Hum Genet. 2014. PMID: 24581741 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases