Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

TY - JOUR

T1 - Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

AU - Johansson, Eva

AU - Fanø, Mathias

AU - Bynck, Julie H

AU - Neuhard, Jan

AU - Larsen, Sine

AU - Sigurskjold, Bent W

AU - Christensen, Ulla

AU - Willemoës, Martin

N1 - Keywords: Alleles; Amino Acid Sequence; Arginine; Binding Sites; Catalysis; DNA Mutational Analysis; Diffusion; Dose-Response Relationship, Drug; Escherichia coli; Genetic Vectors; Glutamic Acid; Hydrolysis; Magnesium; Methanococcus; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutation; Nucleotide Deaminases; Phosphates; Protein Binding; Protein Conformation; Protein Structure, Secondary; Salmonella typhimurium; Selenomethionine; Sequence Homology, Amino Acid; Substrate Specificity; Thymidylate Synthase

PY - 2005

Y1 - 2005

N2 - dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

AB - dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

U2 - 10.1074/jbc.M409534200

DO - 10.1074/jbc.M409534200

M3 - Journal article

C2 - 15539408

SN - 0021-9258

VL - 280

SP - 3051

EP - 3059

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 4

ER -