A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.