The intramolecular autoglucosylation of monomeric glycogenin

Mechanisms of Monomeric and Dimeric Glycogenin Autoglucosylation

“…Mutagenesis, Expression, and Purification-Glycogenin truncated at residue 270 was obtained as described previously (15). Briefly, the cDNA corresponding to the fragment of interest was inserted in-frame in the pET15b vector downstream of the His 6 tag-encoding sequence of the plasmid.…”

“…The reaction kinetics exhibited by a heterodimer formed by two glycogenin mutants, one lacking glucosylating activity and the other without the acceptor Tyr-194 residue, led an intersubunit reaction mechanism being proposed for autoglucosylation (14). We demonstrated previously that monomeric glycogenin, as it is present in solution at submicromolar concentrations, is able to autoglucosylate by an intramolecular mechanism (15). However, the extent of glucopolymerization produced by the monomer was not determined.…”

Conformational plasticity of glycogenin and its maltosaccharide substrate during glycogen biogenesis

“…An hGYG1 dimer can potentially catalyze the glucosylation of two nascent maltosaccharide chains, and both intra- (13,14) and intersubunit (15) mechanisms have been proposed. To investigate this structurally, we cocrystallized hGYG1 WT-mix with Mn 2þ · UDP and determined structures adopting the active state.…”

“…First, substrate-induced conformational changes to shield the active site, often advocated as integral to the GT catalytic mechanism (5, 10), have not been observed for glycogenin to date. In addition, the possibilities of intrasubunit (13,14) and intersubunit (15) glucosylation exist for a glycogenin dimer, as the growing maltosaccharide chain attached to one subunit may theoretically proceed into the active site of its own or adjacent subunit for further glucosylation. This remains a subject of debate and is complicated by a reported monomeric form of glycogenin at low micromolar concentration (13).…”

“…In addition, the possibilities of intrasubunit (13,14) and intersubunit (15) glucosylation exist for a glycogenin dimer, as the growing maltosaccharide chain attached to one subunit may theoretically proceed into the active site of its own or adjacent subunit for further glucosylation. This remains a subject of debate and is complicated by a reported monomeric form of glycogenin at low micromolar concentration (13). Furthermore, the mechanism by which glycogenin catalyzes the glucosylation reaction with retention of the anomeric carbon configuration is unclear, as are the binding modes for the sugar donor and acceptor, the latter also confounded by the nonconventional UDPG configuration in the rGYG1 structure as compared to other GT-A enzymes (6).…”