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  • Voriconazole br Results br Discussion Thus

    2019-10-09


    Results
    Discussion Thus, the biochemical and structural studies of six Maf proteins from different organisms have revealed two subfamilies of new enzymes with the metal-dependent nucleoside triphosphate pyrophosphatase activity against both canonical and noncanonical pyrimidine nucleotides (YhdE-like proteins) or m7GTP (YceF-like proteins) (Table 1). Crystal structures revealed the active sites of Maf proteins and molecular mechanisms of preference for canonical (dTTP and UTP) and modified nucleotides (pseudo-UTP, m5CTP, m5UTP, and m7GTP), as well as a mechanism of enzyme preference for methylated bases. The preference of Maf proteins for modified ribonucleotides in vitro suggests that, in vivo, these enzymes are likely to monitor the ribonucleotide pool and prevent unspecific incorporation of modified bases into cellular RNAs. In some organisms, Maf proteins can function in parallel with the UTPase subfamily of Nudix hydrolases, which have been shown to be active against 5-methyl-UTP (riboTTP) and UTP in vitro (Xu et al., 2003). Currently, over 110 RNA or DNA Voriconazole modifications are known, which are introduced by specific modifying enzymes and are important for gene expression, translation, DNA repair, stress response, and host-pathogen interactions (Cantara et al., 2011). The pseudouridine (Ψ), 5-methyluridine (m5U), 5-methylcytidine (m5C), and 7-methylguanosine (m7G) are the most abundant modified nucleosides found in all organisms, with Ψ being the most abundant modified nucleoside in both prokaryotic and eukaryotic RNAs. They are synthesized at the RNA level by different modifying enzymes including pseudouridine synthases and methylases, which catalyze the site-specific isomerisation or methylation of RNA bases (Gustafsson et al., 1996, Koonin, 1996) (Figure 7). It is expected that degradation and repair of cellular nucleic acids containing modified bases will produce the respective nucleoside monophosphates (m5CMP, m5dCMP, m5UMP, and m7GMP), which can be converted into triphosphates and incorporated into newly synthesized nucleic acids by RNA polymerases (Bessman et al., 1958, Goldberg and Rabinowitz, 1963, Kahan and Hurwitz, 1962) (Figure 7). Thus, hydrolysis of the modified nucleoside triphosphates by Maf proteins in vivo might reduce their incorporation into cellular nucleic acids and diminish their potential mutagenic effects. With canonical nucleotides as substrates, the YhdE-like Maf proteins showed high affinity to dTTP, UTP, and CTP with a micromolar KM (25.0 to 105.9 μM), which is within the range of known intracellular concentrations of these nucleotides in prokaryotic and eukaryotic cells (1.5 μM to 8 mM) (Bennett et al., 2009, Ditzelmüller et al., 1983). Thus, in vivo, Maf proteins can potentially slow down the synthesis of both DNA and RNA. Our metabolome analysis confirmed that dTTP and UTP are the in vivo substrates for the E. coli YhdE and, potentially, for other YhdE-like proteins. We postulate that the pyrophosphatase activity of Maf proteins against the canonical pyrimidine nucleotides (Figure 1) might represent one of the molecular mechanisms of the inhibition of cell division by Maf proteins. This is consistent with the results of recent work on B. subtilis, which revealed that the BSU28050 K53A mutant protein is deficient in the complementation of a maf deletion, as observed by the lack of restoration of the comGA::Tn917 filamentous phenotype (Briley et al., 2011). Our in vitro assays with the purified mutant proteins BSU28050 (K53A), YhdE (K52A), YceF (K52A), YOR111W (K74A), and ASMTL-Maf (K65A) demonstrated complete, or almost complete, inactivation of nucleotide pyrophosphatase activity in all proteins (Figure 4), supporting our hypothesis that this activity plays an important role in the in vivo function of Maf proteins.
    Significance
    Experimental Procedures
    Acknowledgments