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Role of the Two Catalytic Domains of DSR-E Dextransucrase and Their Involvement in the Formation of Highly -1,2 Branched Dextran

Emeline Fabre, Sophie Bozonnet, Audrey Arcache, René-Marc Willemot, Michel Vignon, Pierre Monsan, and Magali Remaud-Simeon^*

Centre de Bioingéniérie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, DGBA INSA, Toulouse, France

Received 21 June 2004/ Accepted 19 September 2004

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both -1,6 and -1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by ¹³C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only -1,6 linkages. On the other hand, enzymes devoid of CD1 modified -1,6 linear oligosaccharides and dextran acceptors through the formation of -1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of -1,6 glucosidic bonds and CD2 only catalyzing the formation of -1,2 linkages. This finding permitted us to elucidate the mechanism of -1,2 branching formation and to engineer a novel transglucosidase specific for the formation of -1,2 linkages. This enzyme will be very useful to control the rate of -1,2 linkage synthesis in dextran or oligosaccharide production.

Present address: BNG-Biocentrum, Danish Technical University, DK-2800 Kgs. Lyngby, Denmark.

Present address: Centre de Recherche sur les Macromolécules Végétales, 38041 Grenoble Cedex 9, France.

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