Levansucrase LevS from Leuconostoc mesenteroides B-512F is a multidomain fructansucrase (MD-FN) that contains additional domains (ADs) to the catalytic domain. However, the understanding of the effect that these ADs have on enzyme activity remains vague. To this aim, structure-function relationship studies of these LevS ADs were performed by evaluating both biochemical properties and the enzymatic capacity of truncated versions of LevS. Joint participation of the N- and C-terminal domains is essential for stability, activity, specificity, and polymerization processes. Specifically, the N-terminal region is involved in stability, while the transition region plays an essential role in the transfructosylation reaction and polymer elongation. Based on our results, we suggest that ADs interact with each other, adopting a U-shaped topology. The importance of these ADs observed in the MD-FN of the Leuconostocaceae family is not shared by the Lactobacillaceae family. Phylogenetic analysis of LevS AD suggests that MD-FN from Lactobacillaceae and Leuconostocaceae have different evolutionary origins. This is the first study on the structure-function relationship of multidomain levansucrases from the Leuconostocaceae family. Our results point towards the functional role of AD in MD-FN and its involvement in fructan synthesis.