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

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Author: Ozimek, L.K. · Euverink, G.J.W. · Maarel, M.J.E.C. · Dijkhuizen, L.
Type:article
Date:2005
Institution: TNO Kwaliteit van Leven TNO Voeding
Source:FEBS Letters, 579, 1124-1128
Identifier: 88666
Keywords: Food technology · Lactobacillus reuteri · Levansucrase · Mutagenesis · Bacterial enzyme · Fructosyltransferase · Inulosucrase · Levansucrase · Metal ion · Unclassified drug · Amino acid sequence · Bacillus subtilis · Bacterial strain · Calcium binding · Calcium transport · Enzyme activity · Enzyme structure · Gram positive bacterium · Metal binding · Mutational analysis · Nonhuman · Nucleotide sequence · Priority journal · Protein family · Site directed mutagenesis · Amino Acid Sequence · Binding Sites · Calcium · Edetic Acid · Enzyme Stability · Hexosyltransferases · Ion Transport · Lactobacillus · Molecular Sequence Data · Mutation · Protein Denaturation · Sequence Alignment · Temperature

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. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Molecular Sequence Numbers: GENBANK: AF459437, AF465251; Chemicals / CAS: calcium ion, 14127-61-8; fructosyltransferase, 9031-67-8; levansucrase, 9030-17-5; Calcium, 7440-70-2; Edetic Acid, 60-00-4; Hexosyltransferases, EC 2.4.1.-; inulosucrase, EC 2.4.1.9; levansucrase, EC 2.4.1.10