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

doi:10.1007/s10930-006-9032-5

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 journal › Article › peer-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 language	English (US)
Pages (from-to)	483-491
Number of pages	9
Journal	Protein Journal
Volume	25
Issue number	7-8
DOIs	https://doi.org/10.1007/s10930-006-9032-5
State	Published - Dec 1 2006

All Science Journal Classification (ASJC) codes

Analytical Chemistry
Bioengineering
Biochemistry
Organic Chemistry

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title = "The effects of removing the GAT domain from E. coli GMP synthetase",

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.",

author = "Abbott, {Jessica L.} and Newell, {Jordan M.} and Lightcap, {Christine M.} and Olanich, {Mary E.} and Loughlin, {Danielle T.} and Weller, {Melanie A.} and Gary Lam and Sidney Pollack and Patton, {Walter A.}",

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journal = "Protein Journal",

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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.

In: Protein Journal, Vol. 25, No. 7-8, 01.12.2006, p. 483-491.

Research output: Contribution to journal › Article › peer-review

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AU - Abbott, Jessica L.

AU - Newell, Jordan M.

AU - Lightcap, Christine M.

AU - Olanich, Mary E.

AU - Loughlin, Danielle T.

AU - Weller, Melanie A.

AU - Lam, Gary

AU - Pollack, Sidney

AU - Patton, Walter A.

PY - 2006/12/1

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N2 - 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.

AB - 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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