Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121
article
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 1.5-4×105 times reduction of total sucrase activity. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Topics
Food technologyCatalytic residueFructosyltransferaseInulosucraseLevansucraseMutagenesisAcidBaseGlycosidaseLevansucraseMutant proteinStabilizing agentSucraseTransferaseBacterium mutantEnzyme activityNonhumanSite directed mutagenesisAmino Acid MotifsAmino Acid SequenceAmino Acid SubstitutionCatalysisCircular DichroismCloning, MolecularConserved SequenceEscherichia coliGene ExpressionGenes, BacterialHexosyltransferasesKineticsLactobacillusMolecular Sequence DataMutagenesis, Site-DirectedSequence Homology, Amino AcidSubstrate SpecificityBacillus subtilisBacteria (microorganisms)Lactobacillus reuteri
TNO Identifier
237623
ISSN
00145793
Source
FEBS Letters, 560(1-3), pp. 131-133.
Pages
131-133