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

Abbott, Jessica L.; Newell, Jordan M.; Lightcap, Christine M.; Olanich, Mary E.; Loughlin, Danielle T.; Weller, Melanie A.; Lam, Gary; Pollack, Sidney; Patton, Walter A.

doi:10.1007/s10930-006-9032-5

Published: 14 November 2006

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

Jessica L. Abbott¹,
Jordan M. Newell²,
Christine M. Lightcap¹,
Mary E. Olanich²,
Danielle T. Loughlin¹,
Melanie A. Weller²,
Gary Lam¹,
Sidney Pollack² &
Walter A. Patton¹

The Protein Journal volume 25, pages 483–491 (2006)Cite this article

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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.

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Abbreviations

ATPP:: ATP-pyrophosphatase
DTT:: dithiothreitol
EDTA:: ethylenediaminetetraacetic acid
GAT:: glutamine amidotransferase
GMP:: guanosine-5′-monophosphate
HPLC:: high performance liquid chromatography
IPTG:: isopropyl-beta-d-thiogalactopyranoside
LB:: Luria–Bertani
PAGE:: polyacrylamide gel electrophoresis
PCR:: polymerase chain reaction
PBS:: Phosphate buffered saline
SDS-PAGE:: sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEC:: size-exclusion chromatography
TBE:: Tris-borate EDTA
XMP:: xanthosine 5′-monophosphate

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Acknowledgments

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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Affiliations

Department of Chemistry, Lebanon Valley College, 101 N. College Ave., Annville, PA, 17003-1400, USA
Jessica L. Abbott, Christine M. Lightcap, Danielle T. Loughlin, Gary Lam & Walter A. Patton
Department of Biology, Lebanon Valley College, 101 N. College Ave., Annville, PA, 17003-1400, USA
Jordan M. Newell, Mary E. Olanich, Melanie A. Weller & Sidney Pollack

Authors

Jessica L. Abbott
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Jordan M. Newell
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Christine M. Lightcap
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Mary E. Olanich
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Danielle T. Loughlin
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Melanie A. Weller
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Gary Lam
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Sidney Pollack
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Walter A. Patton
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Correspondence to Walter A. Patton.

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Abbott, J., Newell, J., Lightcap, C. et al. The Effects of Removing the GAT Domain from E. coli GMP Synthetase. Protein J 25, 483–491 (2006). https://doi.org/10.1007/s10930-006-9032-5

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Published: 14 November 2006
Issue Date: December 2006
DOI: https://doi.org/10.1007/s10930-006-9032-5

Keywords

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