Biochemical characterization and identification of catalytic residues of the thermostable inorganic pyrophosphatase from Thermococcus litoralis

Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of inorganic pyrophosphate (PPi) into orthophosphate (Pi) in the presence of magnesium ions, thereby driving diverse biosynthetic processes. They have been widely applied in in vitro transcription, PCR, and sequencing platforms. Mechanism of family I PPases has been well established, experimental validation of the functional indispensability of conserved catalytic residues in thermophilic archaeal PPases remains limited. In this study, we characterized a thermostable PPase from the archaeon Thermococcus litoralis (TliPPase) through enzymatic assays, phylogenetic analysis, structural modeling, and site-directed mutagenesis. TliPPase retained robust activity under alkaline and high-temperature conditions and effectively relieved PPi accumulation-induced inhibition in PCR systems. Mutational analysis demonstrated that Tyr56, Asp71, and Asp103 are indispensable for enzymatic activity, consistent with their established functional importance in family I PPases. Together, these findings validate the conserved catalytic architecture of a thermophilic PPase and provide a foundation for future studies and engineering of thermophilic PPases.