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Mimicking the activation of the ADP‐glucose pyrophosphorylase from Agrobacterium tumefaciens by site directed mutagenesis
Author(s) -
Alghamdi Mashael,
Hussien Rania,
Patel Hiral,
Dobrzynski Emily,
Iglesias Alberto,
Ballicora Miguel
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb68
Subject(s) - agrobacterium tumefaciens , biochemistry , allosteric regulation , enzyme , site directed mutagenesis , chemistry , mutant , mutagenesis , lyase , active site , stereochemistry , transformation (genetics) , gene
ADP‐glucose pyrophosphorylase is the enzyme that controls the rate limiting step in the biosynthesis of glycogen and starch in bacteria and plants, respectively. In Agrobacterium tumefaciens , this enzyme is allosterically activated by fructose 6‐phosphate and pyruvate, whereas it is inhibited by AMP. In the crystal structure of the enzyme Ser72 interacts with a sulfate hypothesized to occupy the phosphate of the Fru6P allosteric site. To study the role of this residue, we replaced Ser72 by Ala, Cys, and Trp. In addition, we mutated it to Asp, and Glu to mimic the presence of negative charge of the Fru6P. Replacement by Ala did not significantly change the regulatory properties of the enzyme, indicating that the side chain does not play a critical role. However, we observed that S72D, S72E, S72C, and S72W became insensitive to Fru6P. Cys and Trp residues at position 72 remarkably reduced the specific activity of the enzyme (7.4 and 1.7 U/mg, respectively). On the other hand, S72D and S72E were partially activated (22.3 and 18.9 U/mg, respectively, compared to 8.3 U/mg of the WT). This indicates that the introduction of a carboxylate moiety in position 72 hinders the binding of Fru6P, but partially mimics its presence. These mutants were still activated by pyruvate, which indicates that this region is specifically involved in the activation by Fru6P and not by pyruvate. The mutants were not inhibited AMP inhibitor suggesting that the binding site for Fru6P and inhibitor overlaps. Our results suggest that Ser72 is in the Fru6P regulatory site and, consequently, the sulfate found in the crystal structure must be occupying the site of the phosphate moiety. Support or Funding Information This work was supported by NSF Grant MCB 1616851 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .