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. Patton

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)483-491
Number of pages9
JournalProtein Journal
Volume25
Issue number7-8
DOIs
StatePublished - Dec 1 2006

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Bioengineering
  • Biochemistry
  • Organic Chemistry

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