title: Residue Leu940 has a crucial role in the linkage and reaction specificity of the glucansucrase GTF180 of the probiotic bacterium Lactobacillus reuteri 180.
abstract: Highly conserved glycoside hydrolase family 70 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 Leu(940) 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 Leu(940) 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 physicochemical properties (containing 67-100% of (α1→6) linkages) were produced by GTF180 and its Leu(940) 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 Leu(940) 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.
authors.affiliation: From the Departments of Microbial Physiology and.
authors.forename: Xiangfeng
authors.initials: X
authors.lastname: Meng
authors.affiliation: From the Departments of Microbial Physiology and.
authors.forename: Justyna M
authors.initials: JM
authors.lastname: Dobruchowska
authors.affiliation: Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Tjaard
authors.initials: T
authors.lastname: Pijning
authors.affiliation: Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Cesar A
authors.initials: CA
authors.lastname: López
authors.affiliation: From the Departments of Microbial Physiology and.
authors.forename: Johannis P
authors.initials: JP
authors.lastname: Kamerling
authors.affiliation: From the Departments of Microbial Physiology and l.dijkhuizen@rug.nl.
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D018360:Crystallography, X-Ray; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D019936:Probiotics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D009844:Oligosaccharides; D013395:Sucrose; D008320:Maltose; D014364:Tryptophan; D016695:Glycosyltransferases; C033837:alternansucrase; D007930:Leucine; D005947:Glucose
keywords: Enzyme Catalysis; Glucansucrase; Oligosaccharide; Polysaccharide; Protein Engineering; Site-directed Mutagenesis; Specificity; α-Glucan