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4,3-?-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H.

ABSTRACT: Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into ?-glucan polysaccharides with (?1???2)-, (?1???3)-, (?1???4)- and/or (?1???6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-acting enzymes characterized so far possess 4,6-?-glucanotransferase activity, cleaving (?1???4)-linkages and synthesizing new (?1???6)-linkages. In this work a gene encoding a putative GH70 family enzyme was identified in the genome of Lactobacillus fermentum NCC 2970, displaying high sequence identity with L. reuteri 121 GtfB 4,6-?-glucanotransferase, but also with unique variations in some substrate-binding residues of GSs. Characterization of this L. fermentum GtfB and its products revealed that it acts as a 4,3-?-glucanotransferase, converting amylose into a new type of ?-glucan with alternating (?1???3)/(? 1???4)-linkages and with (?1???3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of ?-glucans that can be synthesized and allows the identification of key positions governing the linkage specificity within the active site of the GtfB-like GH70 subfamily of enzymes.

SUBMITTER: Gangoiti J

PROVIDER: S-EPMC5216370 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H.

Gangoiti Joana J van Leeuwen Sander S SS Gerwig Gerrit J GJ Duboux Stéphane S Vafiadi Christina C Pijning Tjaard T Dijkhuizen Lubbert L

Scientific reports 20170106

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-a ...[more]

PMID: 28059108

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Project description:Retaining glycoside hydrolases use acid/base catalysis with an enzymatic acid/base protonating the glycosidic bond oxygen to facilitate leaving-group departure alongside attack by a catalytic nucleophile to form a covalent intermediate. Generally, this acid/base protonates the oxygen laterally with respect to the sugar ring, which places the catalytic acid/base and nucleophile carboxylates within about 4.5-6.5 Å of each other. However, in glycoside hydrolase (GH) family 116, including disease-related human acid β-glucosidase 2 (GBA2), the distance between the catalytic acid/base and the nucleophile is around 8 Å (PDB: 5BVU) and the catalytic acid/base appears to be above the plane of the pyranose ring, rather than being lateral to that plane, which could have catalytic consequences. However, no structure of an enzyme-substrate complex is available for this GH family. Here, we report the structures of Thermoanaerobacterium xylanolyticum β-glucosidase (TxGH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose and its catalytic mechanism. We confirm that the amide hydrogen bonding to the glycosidic oxygen is in a perpendicular rather than lateral orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations of the glycosylation half-reaction in wild-type TxGH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed 4C1 chair at the -1 subsite. Nevertheless, the reaction can still proceed through a 4H3 half-chair transition state, as in classical retaining β-glucosidases, as the catalytic acid D593 protonates the perpendicular electron pair. The glucose C6OH is locked in a gauche, trans orientation with respect to the C5-O5 and C4-C5 bonds to facilitate perpendicular protonation. These data imply a unique protonation trajectory in Clan-O glycoside hydrolases, which has strong implications for the design of inhibitors specific to either lateral protonators, such as human GBA1, or perpendicular protonators, such as human GBA2.

Dataset Information

4,3-?-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H.

Publications

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H.

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