Atomic (0.94 Å) resolution structure of an inverting glycosidase in complex with substrate

Guérin, Diego M. A.; Lascombe, Marie-Bernard; Costabel, Marcelo Daniel; Souchon, H.; Lamzin, Victor S.; Béguin, Pierre; Alzari, Pedro M.

doi:10.1006/jmbi.2001.5404

Cited by 128 publications

(151 citation statements)

References 38 publications

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“…Glycine molecules are bound on the surface mainly through ionic interactions. 501,502 ) are bound in the deep pocket, which was confirmed to be an active site in ␣ 6 /␣ 6 -barrel enzymes, and is formed by residues belonging to inner helices and long loops (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47) (Figs. 2c and 5 atom of its amino group to the Asp 149 O ␦1 atom (2.9 Å).…”

Section: Resultsmentioning

confidence: 94%

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Itoh

Akao

Hashimoto

et al. 2004

Journal of Biological Chemistry

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Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated D-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 Å resolution with a final R-factor of 16.8% for 25 to 1.8 Å resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an ␣ 6 /␣ 6 -barrel, whose structure is found in the six-hairpin enzyme superfamily of an ␣/␣-toroidal fold. One side of the UGL ␣ 6 /␣ 6 -barrel structure consists of long loops containing three short ␤-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp 88 and Asp 149 . This was supported by site-directed mutagenesis studies in Asp 88 and Asp 149 .

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Section: Resultsmentioning

confidence: 94%

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Itoh

Akao

Hashimoto

et al. 2004

Journal of Biological Chemistry

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“…Although the general catalytic mechanism of inverting glycosidases is known, the transition state and the conformations adopted by the pyranosyl ring of the Ϫ1 sugar are still indefinite. Some reports on the inverting GH8 enzymes suggested that the Ϫ1 sugar ring displays an unfavorable boat 2,5 B conformation (42,43). We have been trying to get insight into the structural changes of the Ϫ1 sugar along the reaction pathway based on the crystal structure of the enzyme-NAG oligomer complexes.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of the Catalytic Domain of a Novel Glycohydrolase Family 23 Chitinase from Ralstonia sp. A-471 Reveals a Unique Arrangement of the Catalytic Residues for Inverting Chitin Hydrolysis

Arimori

Kawamoto

Shinya

et al. 2013

Journal of Biological Chemistry

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Background: Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) is a chitinase that was first found in glycohydrolase family 23. Results: The crystal structure of Ra-ChiC exhibited a tunnel-shaped conformation in its active site. Conclusion:The tunnel-shaped conformation is essential for a unique arrangement of the catalytic residues and substrate specificity. Significance: This is the first report on the tunnel-shaped binding site of an inverting chitinase.

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“…28 Guerin and co-workers published a high-resolution crystal structure, PDB 1KWF, of a mutated (E95Q) GH8 endoglucanase (EG) from the C. thermocellum cellulosome (Figure 2). 25 A cellopentaose molecule is bound in a groove-shaped active site, characteristic of EGs, spanning subsites -3, -2, -1, +1, and +2. The active site forces the cellopentaose chain to kink, and it distorts the glucosyl residue in subsite -1 away from the ground-state 4 C 1 conformation to a 2 S O / 2,5 B ring conformation, while the other four glucosyl residues remain in the relaxed 4 C 1 conformation.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

et al. 2009

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A detailed understanding of the catalytic strategy of cellulases is key to finding alternative ways to hydrolyze cellulose to mono-, di-, and oligosaccharides. Endoglucanases from glycoside hydrolase family 8 (GH8) catalyze the hydrolysis of β-1,4-glycosidic bonds in cellulose by an inverting mechanism believed to involve a oxacarbenium ion-like transition state (TS) with a boat-type conformation of the glucosyl unit in subsite −1. In this work, hydrolysis by Clostridium thermocellum endo-1,4-glucanase A was computationally simulated with quantum mechanics/molecular mechanics metadynamics based on density functional theory. Our calculations show that the glucosyl residue in subsite −1 in the Michaelis complex is in a distorted 2 S O / 2,5 B ring conformation, agreeing well with its crystal structure. In addition, our simulations capture the cationic oxacarbenium ion-like character of the TS with a partially formed double bond between the ring oxygen and C5′ carbon atoms. They also provide previously unknown structural information of important states along the reaction pathway. The simulations clearly show for the first time in GH8 members that the TS features a boattype conformation of the glucosyl unit in subsite −1. The overall catalytic mechanism follows a D N *A N -like mechanism and a β-2 S O → 2,5 B [TS] → α-5 S 1 conformational itinerary along the reaction coordinate, consistent with the anti-periplanar lone pair hypothesis. Because of the structural similarities and sequence homology among all GH8 members, our results can be extended to all GH8 cellulases, xylanases, and other endoglucanases. In addition, we provide evidence supporting the role of Asp278 as the catalytic proton acceptor (general base) for GH8a subfamily members. Disciplines Biochemical and Biomolecular Engineering | Biological Engineering | Chemical Engineering CommentsPosted with permission from The Journal of Physical Chemistry B, 113, no. 20 (2009) ReceiVed: December 29, 2008; ReVised Manuscript ReceiVed: March 18, 2009 A detailed understanding of the catalytic strategy of cellulases is key to finding alternative ways to hydrolyze cellulose to mono-, di-, and oligosaccharides. Endoglucanases from glycoside hydrolase family 8 (GH8) catalyze the hydrolysis of -1,4-glycosidic bonds in cellulose by an inverting mechanism believed to involve a oxacarbenium ion-like transition state (TS) with a boat-type conformation of the glucosyl unit in subsite -1. In this work, hydrolysis by Clostridium thermocellum endo-1,4-glucanase A was computationally simulated with quantum mechanics/molecular mechanics metadynamics based on density functional theory. Our calculations show that the glucosyl residue in subsite -1 in the Michaelis complex is in a distortedB ring conformation, agreeing well with its crystal structure. In addition, our simulations capture the cationic oxacarbenium ion-like character of the TS with a partially formed double bond between the ring oxygen and C5′ carbon atoms. They also provide previously unknown structural inf...

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Atomic (0.94 Å) resolution structure of an inverting glycosidase in complex with substrate

Cited by 128 publications

References 38 publications

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Crystal Structures of the Catalytic Domain of a Novel Glycohydrolase Family 23 Chitinase from Ralstonia sp. A-471 Reveals a Unique Arrangement of the Catalytic Residues for Inverting Chitin Hydrolysis

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

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