Designed by: Andrew Hall   Group: iGEM08_Edinburgh   (2008-10-06)

glgC16 (glgC with G336D substitution)

This is the coding sequence of glgC (ADP-glucose pyrophosphorylase) from Escherichia coli JM109 with the substitution G336D. This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Ball, S.G. and Morell, M.K. 2003. From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Annual Reviews in Plant Biology 54, 207-233; Leung, P., Lee, Y.M., Greenberg, E., Esch, K., Boylan, S., and Preiss, J. 1986. Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADP-glucose pyrophsophorylase with altered allosteric properties. Journal of Bacteriology 167, 82-88; Meyer, C.R., Bork, J.A., Nadler, S.N., Yirsa, J. and Preiss, J. 1998. Site-directed mutagenesis of a regulatory site of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 336 in allosteric behaviour. Archives of Biochemistry and Biophysics 351, 152-159)

Sequence and Features

Assembly Compatibility:
  • 10
  • 12
  • 21
    Illegal BamHI site found at 194
  • 23
  • 25
  • 1000

In Silico Validation

2017 Team NYU Abu Dhabi ran an in silico experiment of part BBa_K118016 which is the glgC (ADP-glucose pyrophosphorylase) from E. coli with mutation Gly336Asp. The properties of this mutation were compared to the wild type by running MD simulations using GROMACS with the CHARMM36 forcefield. We ran an energy minimization for 500 ps using the steepest descent minimization algorithm with a stopping condition of maximum force less than 1000 kJ/mol/nm. We found that the energy was minimized in this time frame as shown by the steady convergence of the potential energy (Figure 1).


The minimized structures were solvated with water >12 A away from the protein surface. Potassium and chlorine atoms were added to neutralize the system and mimic experimental conditions of approximately 30 mM concentrations. We equilibrated in two phases, first under an NVT ensemble and then under an NPT ensemble. Both were found to equilibrate within 150 ps as shown in Figures 2 and 3, respectively. These adjusted the volume to 1 bar and temperature to 300K with restraints on the solutes.



The last frame of the NPT equilibration was extracted to start NVT simulations. A short animation of the protein run for 1 ns is attached. RMSD and radius of gyration analyses were run in order to determine the stability of the protein. The RMSD plot (Figure 4) shows the RMSD of the wildtype and G336D backbone does not fluctuate dramatically over the 1000 ps run, indicating that the structure is stable. The backbone of the G336D is overall more stable than the wildtype in the 1000 ps run. The increased stability may facilitate the increased activity of glgC in the absence of its activator observed during experimental tests.


The radius of gyration, which measures a protein’s compactness, indicates that both proteins are stably folded over the 1 ns production run. The plot shows reasonable variation and, along with the RMSD plots, show that both proteins are very stable in explicit solvent solution (Figure 5). "Glgc_gyrate.png"

Analysis of the overlaid proteins showed only slight difference after alignment (Figure 6). Closeup of the mutation site shows that the longer side chain of aspartic acid causes the displacement of a loop due to steric hindrance with Leu318 (Figure 7). This displacement translates through the rest of the protein, accounting for some of the variance in RMSD.

"294px-Glgc_protein.png" "294px-Glgc_close.png"