Ponente
Descripción
Fructooligosaccharides (FOSs) are soluble prebiotic fibbers with proven health-promoting effects in humans and animals. Bacterial levansucrases (EC 2.4.1.10) catalyze fructosyl transfer reactions from the donor substrate sucrose to the natural acceptors water (sucrose hydrolysis), sucrose (FOSs synthesis), and precursor FOSs (polysaccharide formation). Gluconacetobacter diazotrophicus levansucrase (Gd_LsdA) is distinguishable for the synthesis and accumulation of the β-(2→1)-linked FOSs 1-kestotriose and 1,1-kestotetraose, with a consequent low yield of the β-(2→6)-linked polysaccharide levan. In this work, the substrate-interacting amino acid Asn306 was selected as target for saturation mutagenesis aiming to decipher the structural factors involved in regioselectivity. Asn306 interacts with the glucosyl moiety of the acceptor sucrose or the second fructosyl moiety of the acceptor FOS (subsite +2). HPAEC-PAD analysis of mutated Gd_LsdA variants revealed remarkable differences in the output of the fructosyl transfer reaction. The substitution of Asn306 by a positively charged amino acid (Arg or Lys but not His) directed the acceptor sucrose in an orientation that favored the synthesis of 6-kestotriose over 1-kestotriose. The Asn306Arg and Asn306Lys variants mostly yielded β-(2→6)-linked FOSs with degree of polymerization (DP) between 3 and 6 which were no further elongated in prolonged reactions. Our results provide insight into a key structural determinant of the regioselectivity and processivity of the fructosyl transfer reaction in levansucrases.
