Insight of Metal Ions in Enzymatic Synthesis of Levan: The Metal-Binding Loop

Levan is a fructose polymer with unique physicochemical properties and physiological activities, making it useful in various industries. It is biologically synthesized from sucrose by the enzyme levansucrase, which does not require metal ions for its fructosylation activity. However, during the molecular characterization of a <i>Pseudomonas chlororaphis</i> levansucrase (PcLscA), it was observed that several divalent metal ions stimulate fructosylation activity by over 2.5-fold. To understand the structural basis of this action, a 3D model of PcLscA was constructed using the artificial intelligence-based modeling software (RoseTTAFold). This analysis revealed a novel metal-binding loop motif. Interestingly, this newly identified loop motif was also found in six known fructansucrase crystal structures. Furthermore, metal-stimulated levan synthesis was confirmed in the other two levansucrases. By identifying this loop motif, this study suggests an efficient approach for the enzymatic synthesis of levan, which traditionally is not known to require metal ions as activators.