title: 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.
authors.affiliation: Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA; Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, El-Chatby, Alexandria 21526, Egypt.
authors.forename: Hanan M A
authors.initials: HM
authors.lastname: Moustafa
authors.affiliation: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, El-Chatby, Alexandria 21526, Egypt.
authors.forename: Taha I
authors.initials: TI
authors.lastname: Zaghloul
authors.affiliation: Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin Airport Economic Area, Tianjin 300308, China. Electronic address: ypzhang@vt.edu.
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D057075:Enzyme Assays / Q000379:methods; D004795:Enzyme Stability; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006358:Hot Temperature; D010770:Phosphotransferases / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000378:metabolism; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000378:metabolism
chemical_list: D012267:Ribosemonophosphates; C031154:ribose 1-phosphate; C031626:ribose-5-phosphate; D019298:Glucose-6-Phosphate; C016473:glucose-1,6-bisphosphate; D010770:Phosphotransferases; C022375:phosphopentomutase
keywords: Biocatalysis; Enzymatic assay; Phosphopentomutase; Thermophilic enzyme; Thermotoga maritima