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  • br Acknowledgements br This work was supported by grants


    This work was supported by grants from the National Natural Science Foundation of China (Grant No.: 30260088) and the prophase research for National “973” Project of China (Grant No.: 2007CB116203). The DNA sequences of Hbvp1 and the Hbvp1 promoter were deposited in GenBank, and the accession numbers were AY 514019 and EF 198329, respectively. Author and address to whom reprint requests are to be sent: Zeng Rizhong, Rubber Research Institute, CATAS, Danzhou (571737), Hainan, PR China.
    : Three structures of phosphoribosyl-ATP pyrophosphatase HisE, determined at the Mycobacterium tuberculosis () and Northeast () Structural Genomics Consortia, have been deposited recently to PDB (entries 1y6x, 1yvw and 1yxb). Inspection of these structures confirms most of the analysis presented in this paper. The only notable difference is that the two “tight” dimers in HisE tetramer are packed together at a different angle than the structurally similar “tight” dimers of the MazG protein. Introduction Hydrolysis of the α-β phosphodiester bond of ATP and other nucleoside triphosphates (NTPs), followed by hydrolysis of the released pyrophosphate, is a highly exergonic reaction that is often used in cell metabolism to ensure irreversibility of a particular reaction or a pathway. In many instances, pyrophosphorolysis is used as a method of removing non-canonical nucleotide triphosphates, preventing their incorporation into DNA or RNA.1, 2, 3, 4 Known NTP pyrophosphatases belong to several structurally distinct superfamilies, including the Nudix (α+β structure), Maf/HAM1 (α/β), c-Myc tag australia nucleotide α hydrolase (α/β), dUTPase-like (all-β), and alkaline phosphatase-like (α/β) superfamilies.1, 2, 3, 4, 5, 6, 7 These enzymes are typically active only against NTPs, in contrast to the NTP diphosphatases (apyrases, EC, which are active against both NTPs and NDPs, and hydrolyze NTPs to NMPs in two distinct successive phosphate-releasing steps with NDPs as intermediates. Recently, a new dUTP pyrophosphatase was described in the protists Leishmania major and Trypanosoma cruzi.8, 9 This enzyme differs from the classical trimeric dUTPase (EC in a number of features, including the ability to use both dUTP and dUDP as substrates, with dUMP being the product in each case. In contrast to apyrases, the dUTP hydrolysis by this enzyme is accompanied by release of pyrophosphate.8, 10 Structural characterization of two members of this family, the dUTPases from and the gastric pathogenic bacterium Campylobacter jejuni, revealed a homodimer with a novel all-α fold, consisting of 11 α-helices with long connecting loops.11, 12 Sequence comparisons revealed common motifs between the dimeric dUTPases and the dCTPase of enterobacterial phages T2 and T4.8, 13 The latter enzyme, commonly referred to as dCTPase, is active also against dCDP, dUTP and dUDP but does not hydrolyze any other NTP or NDP.14, 15, 16, 17 Further sequence analysis revealed homologs of the all-α dUTPase encoded in the genomes of several Gram-positive bacteria and their phages. This allowed delineation of a “basic module” of the dUTPase/dCTPase family, consisting of just five active site-forming helices. The identification of four acidic residues, which coordinate three phosphate-binding Mg2+ cations, provided a motif with which we could search for more distant sequence relationships of these proteins. We show here that these residues, as well as the key structural elements of the dUTPase/dCTPase family, are conserved in two additional families of nucleoside triphosphate pyrophosphatases (NTP-PPases), namely, phosphoribosyl-ATP pyrophosphatase HisE (EC, an enzyme of histidine biosynthesis, and the apparently non-specific NTP-PPase MazG.19, 20 The suggestion of evolutionary relatedness of these enzymes is confirmed by examination of the crystal structure of the MazG family protein SSO12199 from Sulfolobus solfataricus (PDB entry 1vmg), recently solved at the Joint Center for Structural Genomics†. The dUTPase subunit structure superimposed well with two MazG subunits, suggesting that it has evolved through a duplication of a MazG-like ancestral fold, followed by a loss of active-site residues in the C-terminal MazG-like domain. Furthermore, a comparison to the dUTPase active site allowed us to identify the likely substrate of the Sulfolobus MazG and predict that the numerous enzymes of the MazG family encoded in bacterial, archaeal and eukaryotic genomes play a “house-cleaning” role, purging the cell of non-canonical NTPs and NDPs that might otherwise be incorporated into nascent DNA or c-Myc tag australia RNA.