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


CONCLUSIONS
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.
Del Moral
Sandra
S
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado postal 510-3, C, P, 62250, Cuernavaca, Morelos, México. sandit@ibt.unam.mx
Olvera
Clarita
C
Rodriguez
Maria Elena
ME
Munguia
Agustin Lopez
AL
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D004058:Diffusion; D004492:Edetic Acid; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D005936:Glucans / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000166:cytology / Q000201:enzymology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013696:Temperature; D001426:Bacterial Proteins
D002118:Calcium; D004492:Edetic Acid; D005936:Glucans; D006602:Hexosyltransferases; C021451:inulosucrase; D001426:Bacterial Proteins