Abstract
We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.104 clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide 460DYVHT464 in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity.
Highlights
Bacterial glucansucrases (EC. 2.4.1.) are transglucosidases that synthesize high molecular weight α-glucans, oligosaccharides or glucoconjugates from sucrose, a low cost agroresource, as glucosyl donor
Amino acids presumed to be involved in linkage specificity were first identified by sequence alignment of 37 GS catalytic domains (Figure 1)
Five highly conserved segments emerged from the alignment, three of them corresponding to the signature regions II, III and IV of the GH13 family, and which are close to the catalytic residues
Summary
Bacterial glucansucrases (EC. 2.4.1.) are transglucosidases that synthesize high molecular weight α-glucans, oligosaccharides or glucoconjugates from sucrose, a low cost agroresource, as glucosyl donor. 2.4.1.) are transglucosidases that synthesize high molecular weight α-glucans, oligosaccharides or glucoconjugates from sucrose, a low cost agroresource, as glucosyl donor. Depending on their specificity, glucansucrases (GS) catalyze the formation of both linear and branched α-D-glucans with various types of osidic linkages, namely α(1→2); α(1→3); α(1→4) and/or α(1→6) glucosidic bonds. Glucansucrases (GS) catalyze the formation of both linear and branched α-D-glucans with various types of osidic linkages, namely α(1→2); α(1→3); α(1→4) and/or α(1→6) glucosidic bonds These enzymes are attractive tools for glycodiversification due to their ability to produce carbohydrates of diverse size, structure and physico-chemical properties [1]. These structures were obtained by crystallization of recombinant truncated forms of GS from Lb. reuteri 180 (GTF180-ΔN; PDB: 3LK), S. mutans (GTF-SI; PDB: 3AIE), Ln. mesenteroides NRRL B-1299 (ΔN123-GBD-CD2; PDB: 3TTQ) and Lb. reuteri
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