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

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 ScrK) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 10 s M and 2.7 10 s M, respectively, but also significant ones toward D-tagatose (3.5 10 s M) and the unnatural monosaccharides D-altrose (1.1 10 s M) and D-talose (3.4 10 s M). 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.

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans)

“…[69] The substrates D-mannose, D-fructose, D-psicose, D-altrose, D-tagatose, D-glucose, and D-talose have been regioselectively phosphorylated in the 6-position and isolated yields of 51-77% have been obtained for the corresponding 6-phosphates, demonstrating this mannofructokinase to be a versatile enzyme for efficient phosphorylations of these monosaccharides. [69] Interestingly, this enzyme catalyzed the regioselective phosphorylation of Lsorbose, giving the corresponding L-sorbose-5-phosphate in 67% isolated yield. [69] The availability of suitable enantiocomplementary phosphorylgroup transferring enzymes is of key importance and has facilitated significantly the routes of access to both D-and L-enantiomers of chiral phosphorylated metabolites, as shown in Figure 6 [71] while L-xylulose-1-phosphate is obtained by phosphorylation of L-xylulose catalyzed by L-rhamnulokinase (EC 2.7.1.5).…”

“…[69] Interestingly, this enzyme catalyzed the regioselective phosphorylation of Lsorbose, giving the corresponding L-sorbose-5-phosphate in 67% isolated yield. [69] The availability of suitable enantiocomplementary phosphorylgroup transferring enzymes is of key importance and has facilitated significantly the routes of access to both D-and L-enantiomers of chiral phosphorylated metabolites, as shown in Figure 6 [71] while L-xylulose-1-phosphate is obtained by phosphorylation of L-xylulose catalyzed by L-rhamnulokinase (EC 2.7.1.5). [72] Recombinant ketohexokinase and L-rhamnulokinase RhaB have been used for the selective phosphorylation in the 1-position of a series of (3S)-and (3R)-ketopentoses [73] as well as ketohexoses.…”

“…[ 69 ] The substrates d ‐mannose, d ‐fructose, d ‐psicose, d ‐altrose, d ‐tagatose, d ‐glucose, and d ‐talose have been regioselectively phosphorylated in the 6‐position and isolated yields of 51–77% have been obtained for the corresponding 6‐phosphates, demonstrating this mannofructokinase to be a versatile enzyme for efficient phosphorylations of these monosaccharides. [ 69 ] Interestingly, this enzyme catalyzed the regioselective phosphorylation of l ‐sorbose, giving the corresponding l ‐sorbose‐5‐phosphate in 67% isolated yield. [ 69 ]…”

2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity

“…Rare ketoses have great potential, for instance, as chiral auxiliaries, as sweeteners, or (thanks to their biological properties) in pharmaceutical chemistry [1]. Among them, phosphorylated monosaccharides are of particular interest due to their central role in metabolic pathways [2,3]. Sugar phosphates, having a 2-keto functionality, can be produced by lyases or transferases.…”

“…In vivo, phosphorylated monosaccharides are often obtained by direct phosphorylation of the corresponding monosaccharide, catalyzed by an ATP-dependent kinase. Such enzymes have also been efficiently applied for natural or unusual phosphorylated sugar preparation [2,3]. Kinases, as biocatalysts for the production of rare 2-ketoaldonate-phosphates, could also play a key role in the synthetic design of new biologically interesting compounds and enrich the arsenal of biocatalyst compounds.…”