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.
Highlights
Glycosaminoglycan (GAG)1 side chains of proteoglycans (PGs) are important regulators in a wide range of biological
This paper focuses on the analysis of the specificity of the transfer reaction catalyzed by the human GlcAT-I toward the high energy UDP-glucuronic acid (GlcA) donor substrate
Analysis of the activity of the H308R mutant revealed a dramatic change in the donor substrate specificity, because UDP-Glc, UDP-Man, or UDP-GlcNAc could be efficiently transferred to the digalactose acceptor substrate, in contrast to the wild-type enzyme, which presented a restricted specificity toward UDP-hexuronic acids
Summary
Materials—UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-galacturonic acid (UDP-GalA), UDP-N-acetylglucosamine (UDPGlcNAc), UDP-mannose (UDP-Man), galactosyl-1,3-thiogalactose (Gal-Gal), -glucuronidase (bovine liver), -glucosidase (Caldocellum saccharolyticum), aniline, anti-rabbit alkaline phosphatase-conjugated immunoglobulins, and methanol were from Sigma. In a second set of experiments, the membrane fractions from recombinant yeast cells were treated with 2,3-butanedione (15 mM for 20 min), pelleted by centrifugation, and washed twice in 0.25 M sucrose, 5 mM HEPES buffer (pH 7.4) before activity measurements as above. Docking experiments were conducted following three steps: (i) binding of substrates by tethering UDP part to UDP template of the crystal structure (Protein Data Bank code 1FGG) and relaxing the substrate to minimize global free energy (all backbone and side chains were kept frozen); (ii) tethering off and deleting template UDP to determine lowest energy conformations for the substrate; and (iii) thawing side chains of residues in contact with the substrate for a last energy minimization run, keeping both Gal residues of the acceptor substrate frozen This overall process was repeated 20 independent times for each complex studied
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