Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes.
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.
Ozimek
L K
LK
Centre for Carbohydrate Bioengineering, TNO-University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Euverink
G J W
GJ
van der Maarel
M J E C
MJ
Dijkhuizen
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006602:Hexosyltransferases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D017136:Ion Transport; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation / Q000187:drug effects; D016415:Sequence Alignment; D013696:Temperature; D001426:Bacterial Proteins
D002118:Calcium; D004492:Edetic Acid; D006602:Hexosyltransferases; C021513:levansucrase; C021451:inulosucrase; D001426:Bacterial Proteins