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

Abstract

BackgroundInulosucrase (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.ResultsThrough 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 Ca2+ 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.ConclusionThe 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.

Design of Chimeric Levansucrases with Improved Transglycosylation Activity

“…Additionally, two GW modules also constitute the transitional region between the catalytic and the C-terminal domains in IslA, as described by Olivares-Illana et al (19). Interestingly, removal of this particular region causes IslA to lose its transferase activity, as demonstrated by Del Moral et al (8) through the characterization of truncated IslA constructs. Furthermore, we were also able to identify eight CW motifs (ngWikdn.gnWYYfdsdGkm) in the C-terminal region of MD levansucrases (LevS, LevC, and LevL), which are similar to those found in toxins from Clostridium and Streptococcus spp.…”

“…The GW modules are involved in anchoring the protein to the cell wall through interactions with lipoteichoic acids; each module is independently folded and retains its functionality (4,14). The C-terminal domain of IslA behaves in a similar manner, as demonstrated by direct binding assays (8). Additionally, two GW modules also constitute the transitional region between the catalytic and the C-terminal domains in IslA, as described by Olivares-Illana et al (19).…”

“…While a slight decrease of 10 min in the half-lives of SacB C-LevC and SacB C-DsrP was observed, the half-life of SacB C-IslA increased by 8 min. Del Moral et al (8) report a large decrease in stability when the IslA transitional region is eliminated from the enzyme. However, as SacB is already a stable fructansucrase, it is not surprising that a significant increase in stability did not occur.…”

“…Through the construction of truncated versions of IslA, we have recently demonstrated that the acquired regions in MD-FSs, particularly the C-terminal region, confer new properties to the inulosucrase IslA (8). Structural analysis with intrinsic fluorescence confirmed that the acquired domains in IslA interact with the catalytic domain, resulting in a new conformation that renders the enzyme more stable and generates a switch in specificity from a hydrolytic to a transglycosylase mechanism.…”

“…However, these deletions also caused a 4-fold decrease in stability. Actually, these modifications were evident after deletion of the transitional region, which joins the catalytic and the C-terminal domain, as only slight changes in stability and specificity were observed in the absence of the C-terminal domain (8). As a consequence, because the constructed single domain inulosucrase (IslA4) hydrolyzed 70% of the sucrose, it behaves more like a fructosidase than a fructansucrase.…”