Polynucleotide phosphorylase from esherichia coli: regulation mechanisms and substrate binding

Polynucleotide phosphorylase (PNPase), an enzyme conserved in Bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA releasing nucleotide diphosphates and the reverse polymerisation reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either polyA or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Many issues are still open regarding composition, molecular interactions, assembly pathway, mechanism of action, and physiological significance of this molecular machine. We observed that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase. We also developed investigations aimed at identifying the ATP-binding site of PNPase. By a modeling analysis we tentatively identified residues of two putative ATP-binding sites: Phe77, Phe78, Arg79 in one site; Phe56, Arg93, Phe103 in the other. We replaced by alanine the six residues, but in no case the inhibitory effect was abolished. This indicates that the modeling analysis failed to identify the nucleotide binding site. However, during these studies we also observed that the mutants R79A and F77A displayed reduced polymerization activity using ADP as a substrate, which suggests that the two mutated residues are implicated in the catalytic activity of enzyme. To check this hypothesis we determined the kinetic parameters for ADP, Pi and polyA of our mutants. In the case of R79A, both Km values for ADP and phosphate were much larger than in the wild type (wt), whereas in F77A they were very similar to that of wt. Furthrmore, the kcats of these mutants were of the same order of magnitude of that of the wt. These results suggest that Arg79 but not Phe77 compose the nucleoside diphosphate binding-site. We also determined the Km for polyA to check a possible involvement of the two residues in RNA binding, as previously suggested. Nevertheless, we could not demonstrate significant differences compared to that of the wt, which suggests that either these residues do not interact with RNA, or single mutations are not sufficient to cause a significant decrease in RNA binding. To further characterize the substrate-binding site of PNPase we performed a further modeling analysis using Autodock4. We analyzed in silico wt PNPase and the mutant R79A with bound ADP. In the mutant R79A, the affinity between the enzyme and the substrate was smaller than that of the wt. These data confirm that Arg79 is involved in nucleoside diphosphate binding. The computational analysis also points to an involvement of residues Arg80 and Arg83 in ADP binding. We therefore produced the single mutants R80A and R83A and the double mutant R79/80A. None of the single mutants displayed drastic variations in the kcat compared to the wt, which shows that the mutated residues are not directly implicated in the catalytic process. In the single mutants, the Km for phosphate was much larger than that of the wt, Also R80A displayed a Km for ADP two order of magnitude larger but, in contrast, R83A had a smaller Km. In the double mutant, enzyme activity was very low, which prevented us from performing a reliable determination of the kinetic parameters. The Km for polyA was not significantly changed by the mutations. Based on these results it can be concluded that RNA binding is not compromised by the described mutations and that the relevant residues might be instead involved in phosphate and/or ADP terminal phosphate binding. This conclusion is however conflicting with recent reports that point to a role for Arg79 and Arg80 in RNA binding. Thus, it is likely that the overall pattern of interactions involved in substrate binding and catalysis is far more complex than we initially hypothesized. In particular, it is known that PNPase has the additional RNA binding domains KH and S1: thus, the Km for RNA might not significantly change even though the residues we mutated are actually involved in RNA binding. A better understanding of this issue might be provided either using a shorter RNA substrate, or characterizing a PNPase variant deleted of the KH and S1 domains into which the aforementioned mutations have been introduced, as we are planning to do.