Residue Leu(940) Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Xiangfeng Meng, Justyna M. Dobruchowska, Tjaard Pijning, Cesar Lopez-Bautista, Johannis P. Kamerling, Lubbert Dijkhuizen*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

23 Citaten (Scopus)
153 Downloads (Pure)

Samenvatting

Highly conserved GH70 family glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue L940 in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in L940 of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physico-chemical properties [containing 67% to 100% of (α1→6) linkages] were produced by GTF180 and its L940 mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue L940 in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.

Originele taal-2English
Pagina's (van-tot)32773-32782
Aantal pagina's10
TijdschriftThe Journal of Biological Chemistry
Volume289
Nummer van het tijdschrift47
DOI's
StatusPublished - 21-nov-2014

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