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Title: Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

Journal Article · · Journal of Biological Chemistry
 [1];  [2];  [1];  [3];  [1];  [1];  [1]
  1. National Inst. of Health, Research Triangle Park, NC (United States). National Inst. of Environmental Health Sciences. Genome Integrity and Structural Biology Lab.
  2. Center of Postgraduate Medical Education, Warsaw (Poland). Dept. of Biochemistry and Molecular Biology
  3. Univ. of Bologna (Italy). Dept. of Industrial Chemistry
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The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNA with high affinity (Kd ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent Km for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.

Research Organization:
National Inst. of Health, Research Triangle Park, NC (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Inst. of Health (NIH) (United States)
Contributing Organization:
Center of Postgraduate Medical Education, Warsaw (Poland); Univ. of Bologna (Italy)
Grant/Contract Number:
W-31-109-Eng-38; Z01 ES101905
OSTI ID:
1178833
Journal Information:
Journal of Biological Chemistry, Vol. 290, Issue 16; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 9 works
Citation information provided by
Web of Science

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Allosteric Regulation
Cooperativity
Crystal Structure
DNA-binding Protein
Escherichia coli (E. coli)
dGTPase
Mutator Phenotype