Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

FEBS Lett. 2004 Feb 27;560(1-3):131-3. doi: 10.1016/S0014-5793(04)00085-7.

Abstract

Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935-941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5-4x10(5) times reduction of total sucrase activity.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Catalysis
  • Circular Dichroism
  • Cloning, Molecular
  • Conserved Sequence
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Bacterial
  • Hexosyltransferases / chemistry*
  • Hexosyltransferases / genetics*
  • Hexosyltransferases / isolation & purification
  • Hexosyltransferases / metabolism
  • Kinetics
  • Lactobacillus / enzymology*
  • Lactobacillus / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Hexosyltransferases
  • levansucrase
  • inulosucrase