Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes

Abstract

Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca²⁺ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and Futterer, K. (2003) Nat. Struct. Biol. 10, 935–941] has provided evidence for the presence of a bound metal ion, most likely Ca²⁺. Characterization of site‐directed mutants in the putative Ca²⁺ ion‐binding sites of Lb. reuteri Lev and Inu revealed that the Inu Asp520 and Lev Asp500 residues play an important role in Ca²⁺ binding. Sequence alignments of family GH68 proteins showed that this Ca²⁺ ion‐binding site is (largely) present only in proteins of Gram‐positive origin.