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Levanase

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Last Updated: 12 December 2020

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Levanase

Databases
BRENDABRENDA entry
ExPASyNiceZyme view
IntEnzIntEnz view
KEGGKEGG entry
MetaCycmetabolic pathway
PDB structuresRCSB PDB PDBe PDBsum
PRIAMprofile
Identifiers
CAS number2608508
EC number3.2.1.65

The gut microbiota of healthy adults has two dominating bacterial phyla: Firmicutes and Bacteroidetes 1. Unique feature of Bacteroidetes is their ability to degrade and ferment diverse polysaccharides that allow feeding on dietary fibre-poly-and oligosaccharides not digested by human enzymes 1 2. These bacteria degrade, for example, resistant starch, pectin, galactomannan, glucomannan, arabinogalactan, alginate, laminarin, xylan,-glucan, rhamnogalactan, cellulose and levan 3 4 5. Polysaccharide degradation abilities of Bacteroidetes are encode in specific polysaccharide utilization loci. As a rule, PULs also encode surface-bound endo-acting enzymes that initiate sugar polymer degradation 6. In fructan PUL, this role is fulfil by endo-levanase encode by BT1760 3. Levans,-2 6-link fructose polymers, have currently gain attention as polysaccharides with many medical, food-relate and technological applications 7 8 9 10. Notably,-2 6-link fructo-oligosaccharides resulting from levan hydrolysis were shown to have even higher prebiotic effect on probiotic gut bacteria than commercial-2 1-link I-FOS obtained from inulin degradation 11 12 13 14. As L-FOS are not commercially produce, their prebiotic effects are still poorly study. We have studied biochemical properties of endo-levanase BT1760 and show its very high catalytic activity and ability to produce L-FOS from various levans 15. Thus, BT1760 may have biotechnological application in large-scale production of L-FOS for studies of their physiological effects. As levans are very large and usually branch molecules of molecular weight reaching megadaltons 7 15, this endo-acting enzyme offers great interest from aspect of structural biology of enzymes. Considering endo-acting fructanases, crystal structure is available only for endo-inulinase INU2 of filamentous fungus Aspergillus ficuum 16. With regard to levanases, only one structure was available during our studies: structure of C-terminal-sandwich domain of Bacillus subtilis exo-levanase SacC, founding member of CBM66 family of carbohydrate binding modules 17. Here we introduce the first crystal structures of B. Thetaiotaomicron endo-levanase BT1760. Structure of catalytically active protein was resolved at 1. 65 A, and of its non-catalytic E221A mutant complex with levantetraose at 1. 90. As typical for GH32 family enzymes, BT1760 comprises an N-terminal five-bladed-propeller catalytic domain and a tightly packed C-terminal-sandwich module linked to it through short helix. Substrate-binding pockets of two endo-acting fructanases INU2 and BT1760 were shown to differ in shape and ligand binding mode. Previously, C-terminal domain of BT1760 was characterized as domain of unknown function 15. In light of our results, we suggest that this domain, although structurally similar to carbohydrate binding modules, is rather needed for folding, solubility and stability of the whole protein.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Results

The Bi-modular fold of endo-levanase BT1760 is typical for GH32 family proteins 18 19 20 21 22 23 24 25 26 to which endo-levanase is allocate. The N-terminal domain of BT1760 harbouring catalytic centre has an A-propeller topology of five-sheets. Five propeller blades comprised of antiparallel-strand 1 to 19 and connected by loops of varied length. The C-terminal domain has-sandwich architecture. The N-and C-terminal domains are linked by loop 338-347 aa carrying-helix 339 PDAIDR 344. The N-and C-termini of BT1760 are close to each other.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

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