Processivity of dextransucrases synthesizing very high molar mass dextran is mediated by sugar-binding pockets in domain V.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces the dextran of the highest molar mass reported to date (~10(9) g/mol). In this study, we selected a recombinant form, DSR-OKDelta1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very high molar mass polymer. In the domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of Glycoside-Hydrolase 70 (GH70) glucansucrases and known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKDelta1-Y1162A-F1228A) induced a quasi-complete loss of very high molar mass dextran synthesis, resulting in the production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semi-processive mode of DSR-OKDelta1 towards a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relatively to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role on the control of processivity. A deep structural characterization, if possible with large molar mass sugar ligands, would allow comforting this hypothesis.