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  • br Results and Discussion br


    Results and Discussion
    Conclusions Based on their common NTP pyrophosphatase activity, similar size of the catalytic domains, and the conservation of glutamate residues, it was assumed previously that the MazG and HisE families were related to MutT and other Nudix family hydrolases (see Zhang & Inouye; and the description of domain PF03819 in Pfam). However, comparison of the MutT structure (PDB 1puq) to those of the MazG (PDB 1vmg) and dUTPase (PDB 1w2y) shows that the similarity is limited to the presence of a common ExxE motif, which encompasses active-site glutamate residues and is positioned at the end of a long α-helix. Although this local similarity reflects an analogous catalytic mechanism for the two superfamilies of NTP-pyrophosphatases,12, 62 they possess completely different folds and are not homologous. However, the enzymes of the new all-α NTP-PPase superfamily, with the sole exception of HisE, are predicted to hydrolyze unusual, modified NTPs and NDPs. Thus, albeit structurally distinct from Nudix hydrolases, they probably perform similar, house-cleaning functions, by eliminating abnormal NTPs that arise in the course of cell metabolism, e.g. as a result of oxidative damage. Over-expression of RS21-C6 family proteins in proliferating cells, where accumulation of abnormal NTPs would be most damaging, is compatible with this prediction. We consider a MazG-like subunit as the common ancestor of the family. This has remained as the basic subunit in the MazG line, but has roughly doubled in size through a gene fusion event in the evolution of family members such as the dUTPases. In addition, subsequent to the gene 3902 australia and fusion, one of the two domains has lost the motifs necessary for catalysis and retained only one of the two active sites. While Table 1 lists all the superfamily members that have been found using sequence similarity searches, there remains a possibility that distant members of the same fold might still be uncovered by structural comparisons.
    Methods A complete list of the members of the all-α NTP-PPase superfamily was compiled on the basis of the results of iterative PSI-BLAST searches of the NCBI (Bethesda, MD) non-redundant protein database using the dUTPase sequence and other previously identified superfamily members as queries. Secondary structure predictions for various protein families were performed using several independent methods, including PHD, Predator, PSIPRED, Jpred, and SSPro. The multiple sequence alignment was constructed on the basis of the results of PSI-BLAST, taking into account secondary structure prediction. Each protein family was identified as a tight cluster of homologs with significantly greater similarity to each other than to any other proteins. Structure-based sequence alignments of and dUTPase sequences and the MazG structure (PDB: 1vmg) were used as guides for multiple alignment construction. The structures of the individual subunits of ligand complexes of MazG and dUTPase were superimposed using the protein structure comparison service SSM at the European Bioinformatics Institute† authored by E. Krissinel and K. Henrick. Calculations of accessible surface area were done using the Lee & Richards method with a probe radius 1.4Å.
    The Nudix protein superfamily is vast and diverse , . It comprises 29,400 members in the Pfam database (version 26.0) (), of which less than 1% have been experimentally characterized. Enzymes from this family have been shown to catalyze a large variety of seemingly distantly related reactions, including messenger RNA (mRNA) decapping , alternative mRNA polyadenylation , 3′→5′ RNA exonuclease activity , isopentenyl pyrophosphate isomerization , and hydrolysis of a large group of nucleoside diphosphate derivatives . Other members are involved in ADP-ribose-responsive transcriptional regulation and in the formation of an ADP-ribose-responding calcium channel .