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

Abstract

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 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.

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.

Figure 1

IslA truncated constructions. IslA: complete enzyme; IslA2: deletion of the C-terminal domain; IslA3: deletion of the transition and the C-terminal regions; IslA4: deletion of N/C-terminal region.

Figure 2

Binding assays of native IslA and the truncated form IslA3 with non induced L. citreum CW28 cells. Molecular weight control (M). Gel (a) deals with native IslA (line 1) while gel (b) with IslA3 (line 1). In both gels line 2 refers to the non induced L. citreum CW28 cells; line 3 refers to the washed non induced cells after contact with the protein; and line 4 refers to the protein solution after contact with non induced cells.

Figure 3

Effect of EDTA on native IslA and truncated forms activity. IslA4 (black circles), IslA3 (open circles), IslA2 (black triangles), IslA (open squares). Activity measurements are made after 5 min of incubation with EDTA for the IslA4 and IslA3 forms, and after 180 min for native IslA and IslA2.

Figure 4

Structure modifications of IslA and truncated forms mediated by EDTA and Ca²⁺ ions measured by intrinsic fluorescence (excitation wavelength = 280 nm, emission wavelength = 348 nm) (a) IslA4, 50 μM EDTA and 500 μM Ca²⁺ ions; (b) IslA3, 50 μM EDTA and 500 μM Ca²⁺ ions; (c) IslA2 1000 μM EDTA and 2000 μM Ca²⁺ ions and (d) IslA 5000 μM EDTA and 7000 μM Ca²⁺ ions. EDTA is added after the first fluorescence measurement; time of Ca²⁺ ions addition is indicated by an arrow. For each case, the fluorescence of the heat denaturated protein is shown as a broken line.

Figure 5

Circular Dichroism spectra of native IslA and IslA4 mediated by EDTA and Ca²⁺ ions. (a) IslA4: control (yellow line), IslA4 after incubation for 15 min with 0.5 mM EDTA (brown line); IslA4 after incubation for 15 min of the previous sample after restoring Ca²⁺ ions (1 mM) (green line). (b) IslA: control (yellow line), IslA after incubation for 180 min with 0.5 mM EDTA (brown line); IslA after incubation for 180 min of the previous sample after restoring Ca²⁺ ions (8 mM) (green line).