Discovery and Biochemical Characterization of a Thermostable Glucose-1-phosphate Nucleotidyltransferase from Thermodesulfatator indicus

Title:Discovery and Biochemical Characterization of a Thermostable Glucose-1-phosphate Nucleotidyltransferase from Thermodesulfatator indicus

Volume: 24 Issue: 8

Author(s): Qian Li, Ying-Ying Huang, Louis Patrick Conway, Meng He, Shuang Wei, Kun Huang, Xu-Chu Duan, Sabine L. Flitsch and Josef Voglmeir*

Affiliation:

• Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing,China

Keywords: Glucose-1-phosphate nucleotidyltransferase, Thermodesulfatator indicus, NDP-glucose, nucleotide sugars, enzymatic assay, substrate specificity.

Abstract: Background: The biosynthesis of NDP-glucoses is based on the nucleotide transfer from NTP donor substrates to glucose-1-phosphates catalyzed by glucose-1-phosphate nucleotidyltransferases.

Objectives: The cloning and biochemical characterization of a glucose-1-phosphate nucleotidyltransferase (TiGPNT) from the deep sea bacterium Thermodesulfatator indicus.

Methods: The biochemical parameters of recombinant TiGPNT were determined using a plate reader-based coupled enzymatic assay, in which the reaction product UDP-glucose is oxidized in the presence of NAD+ forming UDP-Glucuronic acid and NADH. The substrate promiscuity of the enzyme was determined using thin-layer chromatography and MALDI-ToF mass spectrometry.

Results: TiGPNT was recombinantly expressed under the control of the T7 promoter in Escherichia coli and could be successfully enriched by heat treatment at 80°C for 30 min. The obtained enzyme worked best at pH 7.5 and the optimum reaction temperature was determined to be 50°C. Interestingly, TiGPNT could fully retain its activity even after extended incubation periods at temperatures of up to 80°C. The enzyme was strongly inhibited in the presence of Cu2+ and Fe2+ ions and EDTA. Among the tested glycosyl donor substrates, TiGPNT showed strict specificity towards glucose-1-phosphate. At the same time, TiGPNT was highly promiscuous towards all tested nucleotide donor substrates.

Conclusion: TiGPNT shows comparable biochemical features in regards to pH optima, temperature optima and the substrate specificity to characterized glucose-1-phosphate nucleotidyltransferase from other species. The enzyme was capable of utilizing glucose-1-phosphate and all tested nucleoside triphosphate donors as substrates. The high activity of the enzyme and the simple purification protocol make TiGPNT an interesting new biocatalyst for the synthesis of glucose-diphospho nucleosides.

Cite this article as:

Li Qian, Huang Ying-Ying, Conway Patrick Louis , He Meng, Wei Shuang , Huang Kun, Duan Xu-Chu , Flitsch L. Sabine and Voglmeir Josef *, Discovery and Biochemical Characterization of a Thermostable Glucose-1-phosphate Nucleotidyltransferase from Thermodesulfatator indicus, Protein & Peptide Letters 2017; 24 (8) . https://dx.doi.org/10.2174/0929866524666170724110408

DOI https://dx.doi.org/10.2174/0929866524666170724110408	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305