Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase

Protein Sci. 2022 Jul;31(7):e4376. doi: 10.1002/pro.4376.


The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.

Keywords: ADP-glucose pyrophosphorylase; allosteric regulation; enzyme engineering; glycogen biosynthesis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Agrobacterium tumefaciens* / genetics
  • Agrobacterium tumefaciens* / metabolism
  • Fructose
  • Glucose-1-Phosphate Adenylyltransferase / chemistry
  • Glucose-1-Phosphate Adenylyltransferase / genetics
  • Glucose-1-Phosphate Adenylyltransferase / metabolism
  • Glycogen / metabolism
  • Kinetics
  • Mutagenesis, Site-Directed
  • Phosphates
  • Serine* / genetics
  • Sulfates


  • Phosphates
  • Sulfates
  • Fructose
  • Serine
  • Glycogen
  • Glucose-1-Phosphate Adenylyltransferase