Functional role of the additional domains in inulosucrase(IslA)from Leuconostoc citreum CW28

摘要

Background:Inulosucrase(IslA)from Leuconostoc citreum CW28 belongs to a new subfamily of multidomain fructosyltransferases(FTFs),containing additional domains from glucosyltransferases. It is not known what the function of the additional domains in this subfamily is. Results:Through construction of truncated versions we demonstrate that the acquired regions are involved in anchoring IslA to the cell wall;they also confer stability to the enzyme,generating a larger structure that affects its kinetic properties and reaction specificity,particularly the hydrolysis and transglycosylase ratio.The accessibility of larger molecules such as EDTA to the catalytic domain(where a Ca~(2+) binding site is located)is also affected as demonstrated by the requirement of 100 times higher EDTA concentrations to inactivate IslA with respect to the smallest truncated form. Conclusion:The C-terminal domain may have been acquired to anchor inulosucrase to the cell surface.Furthermore,the acquired domains in IslA interact with the catalytic core resulting in a new conformation that renders the enzyme more stable and switch the specificity from a hydrolytic to a transglycosylase mechanism.Based on these results,chimeric constructions may become a strategy to stabilize and modulate biocatalysts based on FTF activity.