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Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars
- Vergne-Vaxelaire, Carine, Mariage, Aline, Petit, Jean-Louis, Fossey-Jouenne, Aurélie, Guérard-Hélaine, Christine, Darii, Ekaterina, Debard, Adrien, Nepert, Stessy, Pellouin, Virginie, Lemaire, Marielle, Zaparucha, Anne, Salanoubat, Marcel, de Berardinis, Véronique
- Applied microbiology and biotechnology 2018 v.102 no.13 pp. 5569-5583
- Streptococcus mitis, enzyme activity, fructose, glucose, heat tolerance, mannose, open reading frames, pH, phosphorylation, phosphotransferases (kinases), proteins, tagatose, temperature
- Most of the “repressor, open reading frame, kinase” (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrKₘᵢₜᵢₛ) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 10⁶ s⁻¹ M⁻¹ and 2.7 10⁵ s⁻¹ M⁻¹, respectively, but also significant ones toward D-tagatose (3.5 10² s⁻¹ M⁻¹) and the unnatural monosaccharides D-altrose (1.1 10⁴ s⁻¹ M⁻¹) and D-talose (3.4 10² s⁻¹ M⁻¹). Specific activities measured for all hexoses showed a high stereopreference for D- over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.