TY - JOUR
T1 - Structural basis for recognition and catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jannaschii
AU - Huffman, Joy L.
AU - Li, Hong
AU - White, Robert H.
AU - Tainer, John A.
N1 - Funding Information:
The authors thank Andrew Arvai, David Barondeau, Magnar Bjoras, Doug Daniels, Chris Putnam, Samuel Rigby, and Ottar Sundheim for helpful discussions, technical advice, and critical review of this manuscript. This research was supported by a grant from the National Institutes of Health to J.A.T. (GM46312) and a training grant to J.L.H. (HL07781). We also thank the staff at SSRL and ALS for beamline assistance.
PY - 2003/8/22
Y1 - 2003/8/22
N2 - Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure-function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42Å and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77Å and 2.10Å, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase β barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.
AB - Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure-function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42Å and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77Å and 2.10Å, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase β barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.
KW - Deamination
KW - Feedback inhibition
KW - Nucleotide specificity
KW - dCTP deaminase
KW - dUTPase
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U2 - 10.1016/S0022-2836(03)00789-7
DO - 10.1016/S0022-2836(03)00789-7
M3 - Article
C2 - 12909016
AN - SCOPUS:0041665086
SN - 0022-2836
VL - 331
SP - 885
EP - 896
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -