Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121.
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.
Centre for Protein Technology, TNO-Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
L K
LK
Ozimek
S A F T
SA
van Hijum
G A
GA
van Koningsveld
M J E C
MJ
van Der Maarel
G H
GH
van Geel-Schutten
L
L
Dijkhuizen
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001426:Bacterial Proteins
D006602:Hexosyltransferases; C021513:levansucrase; C021451:inulosucrase; D001426:Bacterial Proteins