The Protein Journal

, Volume 25, Issue 7–8, pp 483–491 | Cite as

The Effects of Removing the GAT Domain from E. coli GMP Synthetase

  • Jessica L. Abbott
  • Jordan M. Newell
  • Christine M. Lightcap
  • Mary E. Olanich
  • Danielle T. Loughlin
  • Melanie A. Weller
  • Gary Lam
  • Sidney Pollack
  • Walter A. PattonEmail author


E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH 4 + as an NH3 donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.


GMP Synthetase glutamine amidotransferase ATP-pyrophosphatase Escherichia coli; molecular cloning kinetics polymerase chain reaction 







ethylenediaminetetraacetic acid


glutamine amidotransferase




high performance liquid chromatography






polyacrylamide gel electrophoresis


polymerase chain reaction


Phosphate buffered saline


sodium dodecyl sulfate polyacrylamide gel electrophoresis


size-exclusion chromatography


Tris-borate EDTA


xanthosine 5′-monophosphate



Portions of this work were supported by a Research Corporation Cottrell College Science Award (Grant # CC5937), a Franklin Research Grant from the American Philosophical Society (2004) and a Council on Undergraduate Summer Undergraduate Research Fellowship (Jordan Newell – 2004).

Portions of this work were also supported by institutional grants to Lebanon Valley College from the Whitaker Foundation (Summer 2002) and Merck-AAAS (Summers 2003–2005).

A SpectraMax® Plus384 Microplate Spectrophotometer used in this work was obtained with funds from a Camille and Henry Dreyfus Scholar/Fellow Program Supplemental Award to Walter A. Patton, PhD.

The authors wish to thank Dr. Owen Moe (Lebanon Valley College) for many useful discussions throughout the course of this work.


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Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Jessica L. Abbott
    • 1
  • Jordan M. Newell
    • 2
  • Christine M. Lightcap
    • 1
  • Mary E. Olanich
    • 2
  • Danielle T. Loughlin
    • 1
  • Melanie A. Weller
    • 2
  • Gary Lam
    • 1
  • Sidney Pollack
    • 2
  • Walter A. Patton
    • 1
    Email author
  1. 1.Department of ChemistryLebanon Valley CollegeAnnvilleUSA
  2. 2.Department of BiologyLebanon Valley CollegeAnnvilleUSA

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