Fig. 5: O-GlcNAcylation enhances the enzymatic activity of UGDH.

A In vitro establishment of UGDH enzymatic reaction (n = 3). PBS group as a blank control, and the enzyme dead mutant UGDH^T325D as a negative control. B Enzymatic activity of UGDH in the presence of OGT co-expression or TMG treatment (50 μM) in HEK293T cells (n = 4). C Comparison of the enzyme activity of UGDH^WT and UGDH^S350A isolated from HEK293T cells in the presence of 50 μM TMG (n = 3). D Comparison of the enzyme activity of UGDH^WT and UGDH^S350A isolated from Escherichia coli (n = 4). E Steady-state kinetics of UGDH^WT and UGDH^S350A isolated from HEK293T for UDP-glucose (n = 3). F The free energy profile as a function of the distance of UDP-glucose (UDP-Glc) from the binding sites, as obtained from the ten one-microsecond simulations. G The representative structure model of O-GlcNAcylated UGDH. The S350-O-GlcNAc and UDP-glucose are depicted in sticks, while their molecular surfaces are illustrated by dots in blue and magenta, respectively. NADH is represented in green sticks, and UGDH is shown in white cartoon form. Data are presented as means ± SEM. Statistical analyses were performed by an unpaired two-tailed Student’s t-test.