The polysaccharide levan is a homopolymer that reaches 104 fructose units and is structured in β-(2→6) linked main chains, with β-(2→1) branch points. The formation of the polysaccharide levan occurs by a processive mechanism, where the enzyme remains attached to the fructosylated product, which favors its continuous elongation without mediating the release of intermediate oligofructans. [1] Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) catalyzes the transfer of the fructosyl unit of sucrose to a variety of natural acceptors including water (hydrolysis), sucrose (fructooligosaccharides synthesis), and fructans (elongation and polymerization). [2] In the absence of the sucrose substrate, LsdA exhibits exolevanase activity, but does not hydrolyze inulin-type fructans. [3]

The three-dimensional structure of LsdA (PDB ID: 1W18) [4] exhibits a β type architecture of 5 single-domain petals, in which each leaf β adopts the classical “W” topology of four β-antiparallel strands. The β-propeller architecture forms a topologically constrained pocket that encapsulates the central cavity, thereby optimizing the spatial orientation and accessibility of the substrate binding site. In this active site, the transfer of the fructosyl unit takes place through a double displacement catalytic mechanism (Ping-Pong) that involves the formation of a covalently linked enzyme-fructosyl intermediate complex. [3], [5] In LsdA, the conserved residues D135, D309 and E401 are responsible for the nucleophilic attack of the donor substrate (ex: sucrose), the transient stabilization of the enzyme-fructosyl intermediate, and the general acid-base catalysis, respectively. [4]

Other available 3D structures of levansucrases are those of Bacillus subtilis (PDB ID: 1OYG) [6], Bacillus megaterium (PDB ID: 3OM2) [7], Erwinia amylovora (PDB ID: 4D47) [8] and Erwinia tasmaniensis (PDB ID: 6FRW). [9] In all of them, like in LsdA, the donor substrate of the fructosyl residue is arranged in a similar position at the base of the active site cavity. However, the fructans produced differ in size distribution. [2], [10] Some authors have suggested that the specificity of levansucrase products is defined by specific structural elements of the hypervariable loops that enclose the central cavity and interact with the donor and/or acceptor substrate of the fructosyl residue. [5], [11]

G. diazotrophicus levansucrase released large amounts of 1-kestotriose [αglu(1,2)βfru(1,2)βfru] and kestotetraose [αglu(1,2)βfru(1,2)βfru(1,2)βfru], which accumulated in the reaction mixture and fructans with higher DP were synthesized during later stages of the reaction. [3] The ability of LsdA to produce, in addition to levan, high levels of fructooligosaccharides 1-kestotriose and kestotetraose [12], makes it commercially attractive. Fructooligosaccharides (FOSs) are in high demand in the functional sugars market due to their marked prebiotic effect in humans and animals. [13], [14], [15] The main limitation of using LsdA to produce FOSs as prebiotics it is that they are synthesized together with the levan polymer, the latter being its main contaminant.

In LsdA, it is unknown which amino acids of the protein sequence are involved in the transfer of the fructose unit from the enzyme-fructosyl covalent intermediate to the acceptor molecules of sucrose (synthesis of FOSs) and fructans (formation of the levan polymer). The identification and modification of these structural determinants will allow LsdA mutated variants with different fructans products. In this study, key residues in the LsdA active site, playing different roles in the fructosylation reactions (synthesis and polymerization), were identified and modified.