Flexibility of truncated and full‐length glucansucrase GTF180 enzymes from Lactobacillus reuteri 180

Abstract

Glucansucrase enzymes synthesize high-molecular-mass extracellular aglucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180-DN from Lactobacillus reuteri 180 (lacking the N-terminal domain) revealed an elongated overall structure with two remote domains (IV and V) extending away from the core. By contrast, a new crystal form of the a-1,6/a-1,3 specific glucansucrase GTF180-DN shows an approximate 120 o rotation of domain V about a hinge located between domains IV and V, giving a much more compact structure than before. Positional variability of domain V in solution is confirmed by small angle X-ray scattering experiments and rigid-body ensemble calculations. In addition, small angle Xray scattering measurements of full-length GTF180 also provide the first structural data for a full-length glucansucrase, showing that the enzyme has an almost symmetric boomerang-like molecular shape, with a bend likely located between domains IV and V. The~700-residue N-terminal domain, which is not present in the crystal structures, extends away from domain V and the catalytic core of the enzyme. We conclude that, as a result of the hinge region, in solution, GTF180-DN (and likely also the full-length GTF180) shows conformational flexibility; this may be a general feature of GH70 glucansucrases.

Modulation of glucan‐enzyme interactions by domain V in GTF‐SI from Streptococcus mutans

“…On the other hand, the full enzyme containing domain V would have a larger glucan‐binding area, thus enabling the stabilization of larger polymer chains. These results are consistent with the experimental observations obtained with the complete and truncated GTF180 enzyme (without the V domain), which revealed that truncation of the enzyme impairs its polysaccharide‐synthesizing ability . Our results are also consistent with a processive enzymatic mechanisms for GTF‐SI, as it generates large‐size glucans without releasing intermediate products.…”

“…The position of domain V shows a shift of about 20 Å in GTFA‐ΔN compared to GTF180‐ΔN, whereas in ΔN123‐GBD‐CD2 of DSR‐E this domain adopts a completely different position close to the catalytic core, resulting in a more compact enzyme structure . The intrinsic flexibility of domain V has been related to the GTF180 functioning, as this domain may facilitate glucan synthesis by bringing the polysaccharide chains toward and away from the catalytic site . Additionally, the flexibility of domain V in GTF180 has been related to the ionic strength conditions under which crystallization takes place, as observed in the case of L. reuteri N‐terminally truncated glucansucrase GTF‐180 in its orthorhombic apo‐form (PDB code 4AYG), in which high ionic strength conditions lead to a more curved conformation of domain V compared to the 3HZ3 model .…”

“…6,[10][11][12] Regarding domain V conformation, the crystal structures of different glucansucrases revealed a large positional variability for this segment, while other domains remain essentially unaltered. 13 The position of domain V shows a shift of about 20 Å in GTFA-ΔN compared to GTF180-ΔN, whereas in ΔN123-GBD-CD2 of DSR-E this domain adopts a completely different position close to the catalytic core, resulting in a more compact enzyme structure. [14][15][16] The intrinsic flexibility of domain V has been related to the GTF180 functioning, as this domain may facilitate glucan synthesis by bringing the polysaccharide chains toward and away from the catalytic site.…”

Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70

“…Domain V, representing the truncated GBD, is the most distant from the active site. This domain adopts variable positions in GTF180-⌬N glucansucrase due to motions around a hinge located between domains IV and V (38). The branching sucrase ⌬N 123 -GBD-CD2 derived from the bi-functional glucansucrase DSR-E from Leuconostoc citreum NRRL B-1299 (formerly Leuconostoc mesenteroides NRRL B-1299) has its domain V interacting with the domain IV and shows a more compact structure than GTF180-⌬N (9).…”

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

“…Purification of the enzyme could easily be achieved by affinity chromatography over Sephadex (cross-linked dextran), but the dextran used for elution could not be completely removed by subsequent diafiltration. Glucansucrases are often described based on amino acid alignments with other glucansucrase as having an Nterminal variable region, followed by a catalytic domain, and a C-terminal glucan-binding domain (Monchois et al 1999); however, structural analyses show that the protein actually contains five domains (A, B, C, IV, and V) that are formed through a U-shape configuration that involves two regions of the polypeptide for each domain, with the exception of domain C (Ito et al 2011;Meng et al 2015;Pijning et al 2014;Vujicic-Zagar et al 2010). Numerous studies have examined glucan-binding of the C-terminal repeat domain (Haas and Banas 2000;Kingston et al 2002;Monchois et al 1998;Shah et al 2004), which likely binds tightly to regions of α(1→6) in a strongly exothermic reaction (Komatsu et al 2011).…”