PhD ceremony Mr. T. Pijning: Just add sugar... Structural studies on glucansucrase and fructansucrase enzymes from lactobacilli

PhD ceremony: Mr. T. Pijning

Dissertation: Just add sugar... Structural studies on glucansucrase and fructansucrase enzymes from lactobacilli

Promotor(s): prof. B.W. Dijkstra, prof. L. Dijkhuizen

Faculty: Mathematics and Natural Sciences

Extracellular polysaccharides produced by lactic acid bacteria have interesting properties for food-, non-food and medical applications. Polysaccharides of the α-glucan or β-fructan type are synthesized by glucansucrase and fructansucrase enzymes, respectively. This thesis describes 3D-structural studies on glucan- and fructansucrases, using X-ray crystallography and small-angle X-ray scattering techniques.

Of the glucansucrase GTF180 from Lactobacillus reuteri 180 a truncated form (GTF180-ΔN) was crystallized to determine the first 3D structure of this class of enzymes. In different crystal forms, one of the five domains surprisingly takes up completely different positions, resulting in either an elongated or a compact conformation. In solution, GTF180-ΔN only adopts an elongated structure; full-length GTF180 is boomerang-shaped with the N-terminal domain extending away from the other domains.

The crystal structure of glucansucrase GTFA-ΔN from Lactobacillus reuteri 121 was determined and compared to that of GTF180-ΔN. The two enzymes share 78% sequence identity but differ in product specificity: GTFA-ΔN synthesizes an α-glucan with α(1->6) and α(1->4) glycosidic linkages, whereas GTF180-ΔN synthesizes α-glucans with α(1->6) and α(1->3) glycosidic linkages. The structure reveals that amino acid residues near the acceptor binding site, in particular the residue following the transition-state stabilizing residue, play a key role in product specificity.

The first crystal structure of a bacterial sucrase synthesizing β(2->1) linked β-fructan oligosaccharides and polymers (inulin), InuJ from Lactobacillus johnsonii NCC 533, was determined. A comparison with sucrases synthesizing β(2->6) linked β-fructans (levan) explains their common substrate specificity. Product specificity is likely determined by acceptor binding subsites further away from the active site.