Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Abstract

Background: Glucansucrases of lactic acid bacteria catalyze the synthesis of a variety of ␣-glucans from sucrose. Results: Mutations of Leu 940 in domain B of GTF180 significantly altered its linkage and reaction specificity. Conclusion: Residue Leu 940 of GTF180 is a critical structural determinant for linkage and reaction specificity. Significance: This study provides novel insights into the structural basis of glucansucrases to facilitate the production of tailor-made ␣-glucans.

Production of isomelezitose from sucrose by engineered glucansucrases

“…Instead, and unexpectedly, it produced isomelezitose in high yields. This contrasts with L940 substitutions in GTF180-ΔN that produced linear isomaltooligosaccharides or very complex oligosaccharide mixtures, none of which were identified as isomelezitose [8].…”

“…All amino acid substitutions in Leu940 resulted in an increased percentage of α(1→6), with a subsequent decrease in α(1→3) linkages. However, L940E and L940F substitutions also significantly shifted reaction specificity from oligosaccharide to polysaccharide synthesis [8]. It is reasonable to assume that their structures are homologous to those shown in Figure 6.…”

“…Previous work with L. reuteri focused on this key leucine amino acid position, L940, which is located in the binding groove adjacent to the acceptor binding site, because it was highly conserved in dextran and mutan producing glucansucrase and alternansucrase; however, reuteran-producing glucansucrases and an α(1→2) branching glucansucrase were found to have phenylalanine in this position [8]. In that study, Meng et al [8] determined that amino acid substitutions in this position with large aromatic side chains partially blocked this binding groove preventing isomaltooligosaccharide acceptors from interacting with the enzyme in an orientation favorable to forming α(1→3) glycosidic linkages.…”

“…In an attempt to increase glucan yields, we subsequently decided to focus on amino acid substitution at leucine 441 with DsrI. The corresponding residue, Leu940, in a Lactobacillus reuteri glucansucrase GTF180-ΔN, which produces a watersoluble dextran, was shown to be involved in acceptor substrate binding and is crucial to linkage specificity and glucan yields with this enzyme [8,28]. All amino acid substitutions in Leu940 resulted in an increased percentage of α(1→6), with a subsequent decrease in α(1→3) linkages.…”

“…A few exceptions among this diverse enzyme group include reuteran-producing glucansucrase (e.g., reuteransucrase GtfA from L. reuteri [29] and the catalytic domain 2 of α(1→2) synthesizing glucansucrases (e.g., DsrE from L. mesenteroides [26], which contain a phenylalanine in this position). Crystal structure of a fragment from GTF180 from Lactobacillus reuteri 180 shows that this conserved amino acid is part of a loop of domain B, which is involved in the binding groove near the acceptor binding site [8]. This equivalent leucine can be identified in most glucansucrases as the second amino acid position in the following protein motif presented in PROSITE pattern format:…”