A Thermostable Class III Phosphoribosyl Pyrophosphate Synthetase from <i>Pyrolobus fumarii</i> 1A: Characterization and Application Potential for Phosphoribosyl Pyrophosphate Biosynthesis

Phosphoribosyl pyrophosphate (PRPP) functions as a central metabolic intermediate, supplying ribose-5-phosphate moieties for the biosynthesis of nucleotides, certain amino acids, and a range of essential cofactors. In this study, a thermostable phosphoribosyl pyrophosphate synthetase (PfPRS) was identified from the hyper thermophilic archaeon Pyrolobus fumarii 1A, a hyper thermophilic archaeon that grows optimally at 90-113 °C. The prs gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Peak catalytic activity of PfPRS was observed at approximately pH 7.5 and 55 °C and retained over 85% of its activity after 2 h of incubation across pH 4.0-10.5. PfPRS exhibited high thermal stability. The enzyme exhibited half-lives of 12 h at 90 °C, 5 h at 95 °C, and 3 h at 100 °C. Among the nucleotides tested as diphosphate donors, PfPRS showed a strong preference for ATP, whereas ADP served as an effective inhibitor. Kinetic analysis revealed K_m values of 35 µM for R5P and 46 µM for ATP, with turnover rates (k_cat) of 71 s^-1 and 56 s^-1. PfPRS was co-immobilized with polyphosphate kinase 2 (DrPPK2) from Deinococcus radiodurans using a cross-linked enzyme aggregate (CLEA) system to enable ATP regeneration and to explore the feasibility of using PfPRS for PRPP biosynthesis.