Abstract
Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities.
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Abbreviations
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- ORF:
-
Open reading frame
- MOPS:
-
3-Morpholinopropanesulfonic acid
- PMI:
-
Phosphomannose isomerase
- Man-1-P GTase:
-
Mannose-1-phosphate guanylyltransferase
- GDP-Man:
-
GDP-mannose
- Sugar-1-P NTase:
-
Sugar-1-phosphate nucleotidylyltransferase
- GlcN-1-P:
-
Glucosamine-1-phosphate
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Acknowledgments
We thank Mrs. Yuki Machida for her excellent technical assistance. This work was partly supported by a special grant from the Protein 3000 projects of the Ministry of Education, Culture, Sports, Science and Technology, and partly by the Institute for Fermentation, Osaka (IFO).
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Communicated by S. Albers.
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Estimated biosynthetic pathway of mannosylglycerate in P. horikoshii. The enzymatic activity catalyzing each reaction is showing by abbreviation. The enzymatic activities shown with bold characters are those analyzed in this work. (DOCX 233 kb)
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Akutsu, Ji., Zhang, Z., Morita, R. et al. Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Extremophiles 19, 1077–1085 (2015). https://doi.org/10.1007/s00792-015-0779-5
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DOI: https://doi.org/10.1007/s00792-015-0779-5