A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima

Abstract

Phosphopentomutase (PPM) catalyzes the interconversion of α-d-(deoxy)-ribose 1-phosphate and α-d-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside phosphorylase for determining PPM activities on d-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl2 (0.1 mM) and 50 μM α-d-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of Km and kcat are 1.2 mM and 185 s−1 at 90 °C, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date.