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  • br Introduction Noncanonical nucleoside triphosphates NTPs

    2019-09-10


    Introduction Noncanonical nucleoside triphosphates (NTPs; such as deoxyinosine triphosphate (dITP), deoxyxanthosine triphosphate (dXTP), 8-oxo-deoxyguanosine triphosphate, and 2-oxo-deoxyadenosine triphosphate are produced from oxidation, deamination, or other modifications of canonical nucleotides.1, 2, 3 DNA or RNA polymerases can incorporate noncanonical (deoxy)NTPs (dNTPs) into nascent DNA or RNA during replication or transcription, resulting in complete blockage of the polymerization reaction or in mispairing with incorrect nucleotides. Removal of noncanonical dNTPs from the cellular nucleotide pool is carried out by “house-cleaning” bifonazole whose function is “to cleanse the cell of potentially deleterious endogenous metabolites and to modulate the accumulation of intermediates in biochemical pathways.”1, 4 Four known families of house-cleaning enzymes include Nudix hydrolases (which hydrolyze various nucleoside diphosphates), trimeric dUTPases (which are specific to dUTP), all α-NTP pyrophosphatases (which degrade deoxyuridine triphosphate (dUTP), deoxyuridine diphosphate (dUDP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), and 2-oxo-deoxyadenosine triphosphate), and ITPases (which degrade dITP and xanthosine triphosphate (XTP)).1, 4, 5 Inosine triphosphate (ITP) is generated by the phosphorylation of inosine monophosphate (IMP), which is a precursor of both AMP and guanosine monophosphate (GMP), whereas dITP can be produced by oxidative deamination of dATP or by reduction of ITP or IDP.1, 2, 3 XTP (dXTP) is formed by oxidative deamination of guanosine triphosphate (GTP) or deoxyguanosine triphosphate (dGTP). If incorporated into DNA, hypoxanthine (from dITP) or xanthine (from dXTP) can be paired with T, C, or A, resulting in potentially deleterious mutations. The incorporation of these noncanonical nucleotides into DNA is prevented by the activity of dITP/XTP pyrophosphatases, which are conserved proteins present in bacteria, archaea, and eukaryotes. Most ITPases belong to the HAM1 family (IPR002637; 682 sequences in databases) named after the hydroxylaminopurine sensitivity protein-1 from yeasts. In Saccharomyces cerevisiae, the HAM1 protein protects against the mutagenic effects of the base analog hydroxylaminopurine, which is a natural product of monoxygenase activity on adenine. Only three HAM1 proteins—ITPA from humans, MJ0226 from Methanococcus jannaschii, and RdgB from Escherichia coli—have been biochemically characterized.7, 8, 9, 10 These proteins showed activity against both canonical and noncanonical nucleotides, but the latter was hydrolyzed 10–100 times more efficiently.8, 9, 10 Although two ITPase structures have already been published,7, 11 the molecular mechanisms of substrate selectivity and catalysis remain obscure. Structural studies with MJ0226 have demonstrated that the substrate base is exposed to the solvent and has no contacts with the protein. The recent crystal structure of the human ITPA complex with ITP revealed an alternative binding mode for the substrate, with the base sandwiched between two conserved phenylalanines and with the phosphates coordinated by the side chains of several lysines. A similar substrate-binding mode was also suggested by in silico substrate-docking experiments with the structure of TM0159, a predicted ITPase. Previous genetic experiments have suggested that E. coli RdgB is the enzyme responsible for the interception of dITP/dXTP, preventing the incorporation of hypoxanthine/xanthine into DNA.12, 13, 14, 15 Recent biochemical studies have identified the presence of ITP/XTP pyrophosphatase activity in RdgB, but have produced controversial results on the substrate affinity of this enzyme., In this work, we present the results of structural, biochemical, and mutagenic studies of E. coli RdgB and propose a model for its catalytic mechanism. We have identified four amino acid residues (Lys13, Glu41, Lys53, and Asp69) that are absolutely required for catalytic activity, and we have solved the crystal structures of RdgB in a free state or in complex with ITP+Ca (a substrate) or IMP (a product).