The Donor Substrate Specificity of the Human β1,3-Glucuronosyltransferase I toward UDP-Glucuronic Acid Is Determined by Two Crucial Histidine and Arginine Residues

Abstract

The human ␤1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Gal␤1,3Gal␤1,4Xyl␤-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.

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

“…Similar results have been obtained from other GTs (Qasba et al, 2005). Nevertheless, no conserved amino acid residues have been identified as general determinants of sugar specificity (Ouzzine et al, 2002;Kubo et al, 2004;Modolo et al, 2007;Yonekura-Sakakibara et al, 2007).…”

A Single Point Mutation Reverses the Donor Specificity of Human Blood Group B-synthesizing Galactosyltransferase

“…The x-ray structure of the mutant showed that there was sufficient room for the enzyme to accommodate the N-acetyl group of the alternate donor. For ␤1-3-glucuronosyltransferase, mutation of histidine 308 to arginine produces an enzyme that can efficiently utilize UDP-Glc, UDP-Man, and UDP-GlcNAc as well as UDP-GlcA donor (21). However, unlike these examples of broadened donor specificity, the P234S mutation in glycosyltransferase B results in a complete reversal of donor saccharide recognition.…”

Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: A strategy to promote cartilage repair

“…GlcAT-I has received much attention because it plays a central role at a branching point common to various GAG chains, and it has been suggested to be rate-limiting in GAG synthesis in Chinese hamster ovary cells (18). We have been deeply involved in structural and functional studies of human GlcAT-I (19)(20)(21). We recently reported that IL-1␤ down-regulates GlcAT-I expression and activity and suggested that this may contribute to the reduced biosynthesis of GAGs observed in chondrocytes after IL-1␤ treatment (22).…”