Abstract
Pseudouridine (Ψ) is an essential building block of synthetic RNA for medical applications, so methods for its efficient production receive increased interest. Reverse reaction of the Ψ-5'-phosphate (ΨMP) C-glycosidase, that is, d-ribose 5-phosphate (Rib5P) + uracil (Ura) → ΨMP + H(2)O, allows for the installment of the core β-C-riboside structure of Ψ in a completely selective and efficiently equilibrium-driven single-step transformation. However, providing the Rib5P substrate is challenging for process development and optimum solutions can vary depending on the specific production tasks considered. Here, we exploited the less known activity of purine/pyrimidine nucleotide 5'-phosphate nucleosidase (PpnN; EC 3.2.2.10) to cleave uridine 5'-phosphate (UMP), a relatively expedient starting material for ΨMP synthesis, under release of Rib5P and Ura. Using linear cascade transformation in two enzymatic steps performed in one pot, we demonstrate rearrangement of UMP into ΨMP (yield: ≥ 95%) and thereby obtain the C-riboside product at the solubility limit (∼1.5 mol/L) in a productivity of 2.9 × 10(2 )g/L/h. We show that a previously reported R341A-Y347A double variant of Escherichia coli PpnN (RY) exhibited ∼5-fold higher specific activity toward UMP, and was ∼12-fold less sensitive to Rib5P inhibition, than the wild-type enzyme. Under conditions of C-glycosidase applied in twofold excess over PpnN to minimize the effect of Rib5P inhibition, cascade reaction with RY compared to wild-type PpnN still gave ∼5-fold enhanced productivity. In summary, we present a new synthetic route to ΨMP via N- to C-glycoside rearrangement of UMP. Compared to earlier approaches of cascade biocatalysis for ΨMP production from uridine or UMP, this new route is streamlined due to the direct release of Rib5P from the UMP substrate catalyzed by PpnN.