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Biochemical and Structural characterization of a new GH-70 enzyme from Leuconostoc citreum NRRL B-1299
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International audience. Glucansucrases (GS) from glycoside hydrolase family 70 (GH70) are a-transglucosylases found in lactic acid bacteria such as Leuconostoc, Lactobacillus, Streptococcus and Weissella sp. They catalyze the polymerization of glucosyl residues from sucrose, an economical and abundant agroresource. Depending on the enzyme specificity, the α-glucans vary in terms of size, types of glucosidic bonds and degree of branching. This structure variety confers to α-glucans diverse properties, making them useful for several applications in food and pharmaceutical industries (Leemhuis et al., 2013). The sequencing of Leuconostoc citreum NRRL B-1299 genome allowed the identification of a new GS called DSR-M (Passerini et al., 2015). Whereas the majority of GS produce glucans of very high molar mass (105 to 108 Da), this enzyme synthesizes only a low molar mass dextran (around 27kDa, from 100 g/L sucrose) composed exclusively of a-(1→6) linkages. Now, by playing with the initial sucrose concentration and/or the reaction temperature, different dextrans of controlled molar mass ranging from 5 to 27 kDa can be produced, directly from sucrose and with excellent yields (Vuillemin et al. 2015). In parallel, we solved the 3D atomic structure of a truncated form of this enzyme comprising domains A, B, C and IV as well as most of the domain V also called Glucan Binding Domain. The crystal structure at 3.8 Å of the inactive mutant DSRM-D2-E715Q in complex with isomaltohexaose (IM6) (Fig. 1.) allowed us to identify new determinants, both near the catalytic site and in the domain V, that play a critical role in the molar masses of the dextran produced (from 1 to 15 kDa).
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