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.