title: Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules.
abstract: 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 study, 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. Our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.
authors.affiliation: From the Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: Deepa
authors.initials: D
authors.lastname: Singh
authors.affiliation: the Department of Biochemistry and Molecular Biology, Center of Postgraduate Medical Education, 01-813 Warsaw, Poland, and.
authors.forename: Damian
authors.initials: D
authors.lastname: Gawel
authors.affiliation: From the Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: Mark
authors.initials: M
authors.lastname: Itsko
authors.affiliation: the Department of Industrial Chemistry, University of Bologna, 40136 Bologna, Italy.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Hochkoeppler
authors.affiliation: From the Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: Juno M
authors.initials: JM
authors.lastname: Krahn
authors.affiliation: From the Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
authors.affiliation: From the Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, schaaper@niehs.nih.gov.
authors.forename: Roel M
authors.initials: RM
authors.lastname: Schaaper
mesh_terms: D000494:Allosteric Regulation; D020134:Catalytic Domain; D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C029603:deoxyguanosine triphosphate; D010744:Phosphoric Monoester Hydrolases
keywords: Allosteric Regulation; Cooperativity; Crystal Structure; DNA-binding Protein; Escherichia coli (E. coli); Mutator Phenotype; dGTPase