Key result
Mutations at K360 and R174 in enterovirus 71 RNA-dependent RNA polymerase reduced catalytic efficiency and altered pH dependency, suggesting their participation in nucleotidyl transfer.
Why the study?
In viral RNA-dependent RNA polymerases, whether and how active site residues participate in the two-proton transfer reaction remained to be clarified.
Structural and enzymology data suggest that residues K360 and R174 in EV71 RdRP participate in nucleotidyl transfer during viral RNA synthesis.
Hypothesis-generating for EV71 antivirals; leaves open whether K360/R174 are druggable targets.
The nucleic acid polymerase-catalyzed nucleotidyl transfer reaction associated with polymerase active site closure is a key step in the nucleotide addition cycle (NAC). Two proton transfer events can occur in such a nucleotidyl transfer: deprotonation of the priming nucleotide 3'-hydroxyl nucleophile and protonation of the pyrophosphate (PPi) leaving group. In viral RNA-dependent RNA polymerases (RdRPs), whether and how active site residues participate in this two-proton transfer reaction remained to be clarified. Here we report a 2.5 Å resolution crystal structure of enterovirus 71 (EV71) RdRP in a catalytically closed pre-chemistry conformation, with a proposed proton donor candidate K360 in close contact with the NTP γ-phosphate. Enzymology data reveal that K360 mutations not only reduce RdRP catalytic efficiency but also alter pH dependency profiles in both elongation and pre-elongation synthesis modes. Interestingly, mutations at R174, an RdRP-invariant residue in motif F, had similar effects with additional impact on the Michaelis constant of NTP (KM,NTP). However, direct participation in protonation was not evident for K360 or R174. Our data suggest that both K360 and R174 participate in nucleotidyl transfer, while their possible roles in acid-base or positional catalysis are discussed in comparison with other classes of nucleic acid polymerases.
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Li et al. (2022) studied this question. K360 and R174 mutations vs. Wild-type RdRP was evaluated on Catalytic efficiency and pH dependency profiles. Mutations at K360 and R174 in enterovirus 71 RNA-dependent RNA polymerase reduced catalytic efficiency and altered pH dependency, suggesting their participation in nucleotidyl transfer.
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