Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.
Human beta1,3-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 Gal beta 1-3Gal beta 1-4Xyl 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 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta 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 Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.
Pedersen
L C
LC
Pharmacogenetic Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Tsuchida
K
K
Kitagawa
H
H
Sugahara
K
K
Darden
T A
TA
Negishi
M
M
D000595:Amino Acid Sequence; D002384:Catalysis; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002809:Chondroitin Sulfates; D014453:Glucuronosyltransferase; D006497:Heparan Sulfate; D011994:Recombinant Proteins; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase