Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 331, Issue 4, Pages 885-896Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/S0022-2836(03)00789-7
Keywords
dCTP deaminase; dUTPase; deamination; nucleotide specificity; feedback inhibition
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Funding
- NHLBI NIH HHS [HL07781] Funding Source: Medline
- NIGMS NIH HHS [GM46312] Funding Source: Medline
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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 1.42 Angstrom and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77 Angstrom and 2.10 Angstrom, 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 beta 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. (C) 2003 Elsevier Ltd. All rights reserved.
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