Heparan/Chondroitin Sulfate Biosynthesis

Abstract

Human β1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Galβ1-3Galβ1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 Å in the presence of the donor substrate product UDP, the catalytic Mn2+ ion, and the acceptor substrate analog Galβ1-3Galβ1-4Xyl. The enzyme is a α/β protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu227, Asp252, and Glu281 dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu281 is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp194, Asp195, Asp196) has direct interaction with the ribose of the UDP molecule as well as with the Mn2+ ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.