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  • As temperatures increase rates of spontaneous deamination al

    2020-06-28

    As temperatures increase, rates of spontaneous deamination also increase such that hyperthermophilic organisms must have efficient mechanisms for dealing with this potential L-Carnitine inner salt hazard.8., 9. The hyperthermophile Methanococcus jannaschii has both a dCTP deaminase (encoded by the gene at locus MJ0430) and a dUTP pyrophosphatase (MJ1102), but interestingly the dCTP deaminase also harbors dUTPase activity.10., 11. We therefore, refer to the product of the MJ0430 gene as DCD-DUT, for dCTP deaminase/dUTPase. Kinetic data have revealed that the dual activities of the DCD-DUT enzyme occur independently at the same active site and that both activities require Mg2+, indicating that DCD-DUT is able to convert dCTP to dUMP directly, without releasing dUTP as an intermediate. This results in a highly efficient means for producing thymidylate precursors without releasing the potentially harmful dUTP intermediate. As a mechanism of feedback inhibition, dTTP is inhibitory to DCD-DUT. dUTPases have attracted much attention due to their ubiquitous occurrence and essential nature, being found in prokaryotes, eukaryotes, archaea, and several viruses.12., 13., 14. In mammalian cells, nuclear and mitochondrial isoforms of dUTPase are expressed from a single gene, with highest levels occurring in undifferentiated proliferating cells, making them an attractive target for drug design. Indeed, several chemotherapeutic drugs, such as 5-fluorouracil and methotrexate, are thought to act by allowing misincorporation of dUTP into DNA. Some retroviruses, such as feline immunodeficiency virus (FIV) and mouse mammary tumor virus (MMTV), encode their own dUTPases to protect their cDNA from uracil misincorporation during its synthesis in the cytosol.16., 17. Thus, dUTPases may be important targets for rational drug design experiments with a wide range of applications. Primary sequence analyses and phylogenetic tree constructions have revealed that dCTP deaminases and dUTPases together constitute an enzyme superfamily.12., 13., 18. No homology has been detected between the dCTP deaminases and the cytidine or L-Carnitine inner salt deaminases of non-archaeal organisms. Furthermore, Escherichia coli have separate dCTP, cytosine, and cytidine deaminases, as well as a dUTPase.5., 19., 20. Thus, several key questions remain regarding the relationship of bacterial and archaeal dCTP deaminases with the ubiquitous dUTPases, particularly in the case of the archaeal DCD-DUT enzyme, which contains both activities. Crystal structures have been reported for human, , FIV, and equine infectious anaemia virus (EIAV) dUTPases, revealing a trimeric assembly with contributions from each subunit to each of three identical active sites.2., 14., 21., 22., 23., 24. No crystal structures have yet been described for any of the dCTP deaminases, but it has been speculated that they might have a similar overall tertiary and quaternary structure to that of the dUTPases. In all but one of the dUTPase structures, main-chain residues and a structural water molecule are employed to mimic base-pair hydrogen bonding in selecting for dUTP rather than dCTP.2., 14., 23., 24. How these backbone interactions might be altered to accommodate dCTP binding has been unclear, as are the mechanism of dCTP deamination and the residues involved. dTTP has been shown to inhibit the deaminase and pyrophosphatase activities of DCD-DUT, but it is unclear whether the base can be accommodated in the active site, if poor positioning leads to competitive inhibition, or if inhibition proceeds via an alternate route. To address these questions, we have determined the first structure of a dCTP deaminase, DCD-DUT, and we show that, while the sequence is most similar to the dCTP deaminases, the overall fold and subunit assembly are similar to those of the dUTPases. Interestingly, several critical insertions and amino acid replacements confer specificity for dCTP and catalysis of its deamination and provide a platform for dimerization of trimers to form functional hexamers. We have also modeled nucleotides into weak density from cocrystals and compared the resulting structures to docked enzyme-nucleotide complexes to gain insight into how each nucleotide is specifically recognized and bound. These structures allow the first three-dimensional comparison of dUTPase and dCTP deaminase active sites and suggest a mechanism for dCTP deamination catalysis as well as for dTTP inhibition of DCD-DUT activity.