Nucleotide analogs and molecular modeling studies reveal key interactions involved in substrate recognition by the yeast RNA triphosphatase

Abstract

RNA triphosphatases (RTPases) are involved in the addition of the distinctive cap structure found at the 5′ ends of eukaryotic mRNAs. Fungi, protozoa and some DNA viruses possess an RTPase that belongs to the triphosphate tunnel metalloenzyme family of enzymes that can also hydrolyze nucleoside triphosphates. Previous crystallization studies revealed that the phosphohydrolase catalytic core is located in a hydrophilic tunnel composed of antiparallel β-strands. However, all past efforts to obtain structural information on the interaction between RTPases and their substrates were unsuccessful. In the present study, we used computational molecular docking to model the binding of a nucleotide substrate into the yeast RTPase active site. In order to confirm the docking model and to gain additional insights into the molecular determinants involved in substrate recognition, we also evaluated both the phosphohydrolysis and the inhibitory potential of an important number of nucleotide analogs. Our study highlights the importance of specific amino acids for the binding of the sugar, base and triphosphate moieties of the nucleotide substrate, and reveals both the structural flexibility and complexity of the active site. These data illustrate the functional features required for the interaction of an RTPase with a ligand and pave the way to the use of nucleotide analogs as potential inhibitors of RTPases of pathogenic importance.

A Specific Inorganic Triphosphatase from Nitrosomonas europaea

“…On the basis that all known CYTH proteins hydrolyze triphosphates and that PPP i is the simplest triphosphate compound conceivable, PPPase activity might well be the primitive enzymatic activity in the CYTH protein family, which later evolved to hydrolyze more complex organic substrates such as RNA or ThTP in eukaryotes. It should be pointed out that a high affinity for PPP i was already demonstrated in the case of yeast RNA triphosphatases, where PPP i is a potent competitive inhibitor, albeit a poor substrate (29,42,43).…”

The viral RNA capping machinery as a target for antiviral drugs

“…The sulfate is thought to indicate the position of the c-phosphate of the newly synthesized mRNA. In a docking/modelingbased study, a series of nucleoside analogues (6-chloropurine-riboside-5 0 -triphosphate, 6-methylthioguanosine-5 0 -triphosphate, or 8-iodo-guanosine-5 0 -triphosphate) was identified with high affinity for binding and resistant to hydrolysis (Despins et al, 2010;Issur et al, 2009a).…”

The RNA capping machinery as an anti‐infective target

“…Recent studies on the RTases of Saccharomyces cerevisiae (Cet1) and West Nile virus (NS3) have highlighted a number of synthetic purine analogs (6-chloropurine-riboside-5triphosphate, 6-methylthioguanosine-5 -triphosphate, and 8-iodo-guanosine-5 -triphosphate) that harbor a high affinity for the active site of the enzymes while being poorly hydrolyzed in comparison with natural nucleotides. 8,9 Those compounds harbor very interesting inhibition constants in the sub to low micromolar range (0.4-11 µM) for the Cet1 NTPase activity. As the human capping enzyme is structurally and mechanistically different, namely by its inability to hydrolyze free nucleotides, it is tempting to speculate that those nucleotide analogs will not inhibit the RTase activity in the human capping enzyme.…”