title: The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues.
abstract: The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) 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 His(308) 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 Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) 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 His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate.
authors: UMR 7561 CNRS, Université Henri Poincaré Nancy 1, Faculté de Médecine, 54505 Vandoeuvre-lès-Nancy, France. ouzzine@pharmaco-med.u-nancy.fr
authors: Mohamed
authors: M
authors: Ouzzine
authors: Sandrine
authors: S
authors: Gulberti
authors: Nicolas
authors: N
authors: Levoin
authors: Patrick
authors: P
authors: Netter
authors: Jacques
authors: J
authors: Magdalou
authors: Sylvie
authors: S
authors: Fournel-Gigleux
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004852:Epoxy Compounds / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004852:Epoxy Compounds; D011994:Recombinant Proteins; D014535:Uridine Diphosphate Glucuronic Acid; D006639:Histidine; C007366:diepoxybutane; D001120:Arginine; D014453:Glucuronosyltransferase