Just add sugar... Structural studies on Glucansucrase and Fructansucrase enzymes from lactobacilli
PhD ceremony: | dr. ing. T. (Tjaard) Pijning |
When: | January 10, 2014 |
Start: | 14:30 |
Supervisors: | prof. dr. B.W. Dijkstra, L. (Lubbert) Dijkhuizen |
Where: | Academy building RUG |
Faculty: | Science and Engineering |
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- Δ N) 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- Δ N 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- Δ N from
Lactobacillus reuteri 121 was determined and compared to that of
GTF180- Δ N. The two enzymes share 78% sequence identity but
differ in product specificity: GTFA- Δ N synthesizes an α
-glucan with α (1->6) and α (1->4) glycosidic
linkages, whereas GTF180- Δ N 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.