Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture

The recently published human genome with its relatively modest number of ge nes has highlighted the importance of post-transcriptional and post-transla tional modifications, such as alternative splicing or glycosylation, in gen erating the complexities of human biology. The human UDP-N-acetylglucosamin e (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPG1cNAc, an essential precursor for protein glycosylation. To better unde rstand the catalytic mechanism of these enzymes and the role of the alterna tively spliced segment, we have solved the crystal structures of AGX1 and A GX2 in complexes with UDPG1cNAc (at 1.9 and 2.4 Angstrom. resolution, respe ctively) and UDPGaI1Ac (at 2.2 and 2.3 Angstrom resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new member s of the SpsA-GnT I Core superfamily and, together with mutagenesis analysi s, helps identify residues critical for catalysis. Most importantly, our co mbined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a significant modification of the activ e site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties.