The donor substrate specificity beta 1,3-glucuronosyltransferase I toward UDP-glucoronic acid is determined by two crucial histidine and arginine residues

Mohamed Ouzzine, Sandrine Gulberti, Nicolas Levoin, Patrick Netter, Jacques Magdalou, Sylvie Fournel-Gigleux

    Research output: Contribution to journalArticle

    40 Citations (Scopus)

    Abstract

    The human ß1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galß1,3Galß1,4Xylß-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His308 and Arg277 residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His308 by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg277, site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg277 with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His308 and Arg277 residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate
    Original languageEnglish
    Pages (from-to)25439-25445
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume277
    Issue number28
    DOIs
    Publication statusPublished - 2002

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