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

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.