Crystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis.
EXTL2, an alpha1,4-N-acetylhexosaminyltransferase, catalyzes the transfer reaction of N-acetylglucosamine and N-acetylgalactosamine from the respective UDP-sugars to the non-reducing end of [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol, an acceptor substrate analog of the natural common linker of various glycosylaminoglycans. We have solved the x-ray crystal structure of the catalytic domain of mouse EXTL2 in the apo-form and with donor substrates UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol has been determined. These structures reveal three highly conserved residues, Asn-243, Asp-246, and Arg-293, located at the active site. Mutation of these residues greatly decreases the activity. In the ternary complex, an interaction exists between the beta-phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may play a functional role in catalysis. These structures represent the first structures from the exostosin gene family and provide important insight into the mechanisms of alpha1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.
Pedersen
Lars C
LC
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA.
Dong
Jian
J
Taniguchi
Fumiyasu
F
Kitagawa
Hiroshi
H
Krahn
Joe M
JM
Pedersen
Lee G
LG
Sugahara
Kazuyuki
K
Negishi
Masahiko
M
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D019556:COS Cells; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry; D006860:Hydrogen Bonding; D008565:Membrane Proteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D001120:Arginine; D001216:Asparagine; D001224:Aspartic Acid; D006497:Heparan Sulfate; D008565:Membrane Proteins; D017350:N-Acetylgalactosaminyltransferases; D017351:N-Acetylglucosaminyltransferases; D014537:Uridine Diphosphate N-Acetylglucosamine; C110834:EXTL2 protein, human; C023217:UDP-N-acetylgalactosamine (GlcUA-GalNAc-4-sulfate)(4) N-acetylgalactosaminyltransferase