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Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars.

Applied microbiology and biotechnology (2018-05-08)
Carine Vergne-Vaxelaire, Aline Mariage, Jean-Louis Petit, Aurélie Fossey-Jouenne, Christine Guérard-Hélaine, Ekaterina Darii, Adrien Debard, Stessy Nepert, Virginie Pellouin, Marielle Lemaire, Anne Zaparucha, Marcel Salanoubat, Véronique de Berardinis
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
D-Psicose, ≥95%
Sigma-Aldrich
Phospho(enol)pyruvic acid trisodium salt hydrate, ≥97% (enzymatic)
Sigma-Aldrich
D-Allose, 98%
Sigma-Aldrich
L-(+)-Fructose, ≥97.0% (HPLC)
Sigma-Aldrich
D-(−)-Lyxose, 99%
Sigma-Aldrich
L-(+)-Erythrulose, ≥85% (HPLC)
Sigma-Aldrich
D-Xylulose, ≥98% (HPLC), syrup
Sigma-Aldrich
L-(+)-Gulose, ≥98.0% (HPLC)
Sigma-Aldrich
D-(+)-Sorbose, ≥99%
Sigma-Aldrich
D-(+)-Talose, ≥99.0% (HPLC)
Sigma-Aldrich
D-Altrose, ≥97.0% (HPLC)
Sigma-Aldrich
L(+)-Erythrose, ~40%, syrup