Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1.
The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.
Kakuda
Shinako
S
Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Shiba
Tomoo
T
Ishiguro
Masji
M
Tagawa
Hideki
H
Oka
Shogo
S
Kajihara
Yasuhiro
Y
Kawasaki
Toshisuke
T
Wakatsuki
Soichi
S
Kato
Ryuichi
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000378:metabolism; D001665:Binding Sites; D018959:CD57 Antigens / Q000096:biosynthesis* / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D002448:Cell Adhesion; D018360:Crystallography, X-Ray; D019281:Dimerization; D004583:Electrons; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000737:chemistry*; D006801:Humans; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000737:chemistry
D000606:Amino Sugars; D018959:CD57 Antigens; D002241:Carbohydrates; D000939:Epitopes; C000458:N-acetyllactosamine; D008345:Manganese; D014530:Uridine Diphosphate; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase