Related Experiment Video
Updated: Jul 11, 2026
In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity
Published on: January 5, 2016
A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga
Hanan M A Moustafa1, Taha I Zaghloul2, Y-H Percival Zhang3
1Department 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.
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.
Insights
A novel enzymatic assay enables the study of phosphopentomutase (PPM) activity across a wide temperature range. The most active and thermostable PPM enzyme to date was identified from Thermotoga maritima.
Area of Science:
- Biochemistry
- Enzymology
- Thermophilic enzymes
Background:
- Phosphopentomutase (PPM) is crucial for interconverting α-D-(deoxy)-ribose 1-phosphate and α-D-(deoxy)-ribose 5-phosphate.
- Characterizing PPM activity, especially from thermophilic organisms, requires robust and sensitive assays.
Purpose of the Study:
- To develop a versatile enzymatic assay for determining PPM activity.
- To identify and characterize a novel, highly active, and thermostable PPM from Thermotoga maritima.
Main Methods:
- Development of a coupled/uncoupled enzymatic assay using nucleoside phosphorylase for PPM activity measurement.
- Cloning and overexpression of the putative PPM gene (TM0167) from Thermotoga maritima in E. coli.
- Enzyme characterization including optimal temperature, cofactor requirements, kinetic parameters, and thermostability.
Main Results:
- A simple, sensitive, and cost-effective assay for PPM activity was established, functional from 30 to 90 °C.
- Recombinant PPM from T. maritima exhibited maximal activity at 90 °C.
- The enzyme demonstrated high thermostability with a half-life of 156 minutes at 90 °C, and kinetic parameters (Km=1.2 mM, kcat=185 s⁻¹) were determined at this temperature.
Conclusions:
- The developed assay is effective for studying PPM across a broad temperature spectrum.
- The identified TM0167 enzyme represents the most active and thermostable phosphopentomutase reported to date, with significant potential for industrial applications.
