Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1

Abstract

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 (Galβ1-4GlcNAc) but not lacto-N-biose (Galβ1-3GlcNAc) 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 Mn2+, and an acceptor substrate analogue N-acetyllactosamine (Galβ1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an α/β 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 (Asp195-Asp196-Asp197). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val320 and Asn 321, which are located on the C-terminal long loop from a neighboring molecule, and Phe245 contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.