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Truncation of domain V of the multidomain glucansucrase GTF180 of Lactobacillus reuteri 180 heavily impairs its polysaccharide-synthesizing ability.

Applied microbiology and biotechnology (2015-01-15)
Xiangfeng Meng, Justyna M Dobruchowska, Tjaard Pijning, Gerrit J Gerwig, Johannis P Kamerling, Lubbert Dijkhuizen
ABSTRACT

Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide chain follows a U shape course to form five domains, including domains A, B, and C, which resemble those of family GH13 enzymes, and two extra and novel domains (domains IV and V), which are attached to the catalytic core. To elucidate the functional roles of domain V, we have deleted the domain V fragments from both the N- and C-terminal ends (GTF180-ΔNΔV). Truncation of domain V of GTF180-ΔN yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. Instead, GTF180-ΔNΔV produced a large amount of oligosaccharides. Domain V is not involved in determining the linkage specificity, and the size of polysaccharide produced as the polysaccharide produced by GTF180-ΔNΔV was identical in size and structure with that of GTF180-ΔN. The data indicates that GTF180-ΔNΔV acts nonprocessively, frequently initiating synthesis of a new oligosaccharide from sucrose, instead of continuing the synthesis of a full size polysaccharide. Mutations L940E and L940F in GTF180-ΔNΔV, which are involved in the acceptor substrate binding, restored polysaccharide synthesis almost to the level of GTF180-ΔN. These results demonstrated that interactions of growing glucan chains with both domain V and acceptor substrate binding sites are important for polysaccharide synthesis.

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