**1. Introduction**

In the last decade, interest has grown towards levan/inulin oligosaccharides. They have a wide range of applications, from personal care to packaging. These molecules are especially relevant for their medical applications and prebiotic activity [1–5].

Levansucrases (LSCs, EC: 2.4.1.10) and inulosucrases (INUs, EC: 2.4.1.9) are major fructosyltransferases employed as biocatalysts in the synthesis of fructans and fructooligosaccharides (FOSs). Both are members of glycosyl hydrolase family 68 (GH68) [6]. LSC catalyzes the transfructosylation of the fructose component of sucrose by using a variety of acceptor molecules, forming β-(2,6)-linked oligofructans. When a water molecule acts as an acceptor, the reaction results in the hydrolysis of sucrose into glucose and fructose [1].

LSCs are used in the fermentative production of microbial oligosaccharides and polysaccharides due to their ability to interact with low-cost substitutes of sucrose, e.g., syrups and molasses [7,8].

The existence of a wide spectrum of nonconventional fructosyl acceptors explains the biotechnological interest in LSCs. These enzymes can interact with nonconventional fructosyl acceptors and donors [9], such as monosaccharides, disaccharides, and sucrose homologs. For example, LSCs from *Pseudomonas syringae* pv. tomato DC3000 and *Pseudomonas aurantiaca* are able to transfructosylate deoxy sugars or alditols such as fucose, ribose, sorbitol, and xylitol [10]. Among nonconventional substrates, lactose has been one of the most extensively studied. This is due to its combined role with sucrose in a reaction catalyzed by LSCs from *Bacillus spp.* (*B. methylotrophicus* SK21.002 [11], *B. subtilis* NCIMB 11871 [12], and *B. licheniformis* [13]) to produce lactosucrose, which is a trisaccharide with prebiotic activity [14].

Aromatic alcohols such as phenol derivatives (e.g., hydroquinone) [15] and isoflavones (e.g., puerarin) [16] can also be transfructosylated by LSCs from *B. subtilis* (SacB, BsLSC) and *Gluconacetobacter diazotrophicus* (GdLSC), respectively. The improved physical, chemical, and bioactive properties (solubility, stability, availability, and activity) of these glycosides make them relevant to pharmaceutical applications.

In the last decade, several studies have been carried out to understand which residues are the most relevant in the reaction mechanism [2]. Thanks to these studies, engineered glycosyltransferases can be used to obtain specific compounds such as FOSs (e.g., 6-nystose) instead of high-molecular weight (HMW) levan [17].

To better describe the relevant residues, the active site of LSC is commonly divided into layers. Moving from the sucrose binding site outwards, there are three layers: the first, second, and third. Mutations S173A, S173G, and S422A (first layer) in the LSC of *Bacillus megaterium* (BmLSC, PDB ID: 3OM7) increase transfructosylate activity by 194%, 53%, and 42%, respectively [18]. In *P. syringae* pv. tomato, LSC3 with the E146Q mutation (second layer) exhibits increased production of FOSs compared to wildtype [19]. SacB of *B. subtilis* with a Y429N mutation (second layer) has mostly hydrolytic activity and can produce short-chain FOSs instead of HMW levan [20].

Due to the high-yield production of FOSs, the product spectrum and well-optimized production of recombinant enzyme in *Escherichia coli*, LSCs from *Erwinia tasmaniensis* (EtLSC) [21] and *Erwinia amylovora* (EaLSC) [22,23] are interesting candidates for engineered LSCs that produce tailor-made fructans.

The structure of LSC is known in *B. subtilis*, *B. megaterium*, *E. amylovora*, *E. tasmaniensis*, and *G. diazotrophicus*. LSCs have similar structures, and their active sites possess common structural features [24], such as the triad of amino acids involved in catalysis (Asp46, Asp203, and Glu287 in EaLSC) [25]. LSC has been successfully crystallized in complex with sucrose (*B. subtilis*, Protein Data Bank (PDB) ID: 1PT2), raffinose (*B. subtilis*, PDB ID: 3BYN), fructose, and glucose (*E. amylovora,* PDB ID: 4D47). While the sucrose binding site is conserved, superficial areas and volumes vary across species due to variability in the surrounding loops [2,25].

In this report, we present the first known crystal structure of an LSC, EtLSC, in complex with levanbiose (LBS). LBS is an intermediate in the synthesis of oligolevans in the LSC enzyme. The complex was obtained by soaking EtLSC apo crystals (PDB ID: 6FRW) in a concentrated solution of sucrose (0.5 M) in order to trap reaction intermediates/products in the crystals. We describe an unexplored plausible binding site for LBS. We analyzed conserved amino acids in the binding pocket of LBS and compared their structural arrangement to other LSCs from Gram-positive and -negative bacteria. The aims of these analyses were to understand the biological relevance of the binding pocket and explore possible implications for LSC engineering.
