Three-dimensional structure of a thermostable bacterial cellulase

Juy, Michel; Amrt, Adolfo G.; Alzari, P.M.; Poljak, R J; Claeyssens, Marc; Béguin, Pierre; Aubert, Jean-Paul

doi:10.1038/357089a0

Cited by 227 publications

(134 citation statements)

References 24 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: 99%

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: 99%

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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“…Families G and H were added later and a recent comparison of more than 120 sequences allowed the definition of three more families (I-K) resulting in a total of 11 families of cellulases and xylanases (Henrissat and Bairoch, 1993). The three-dimensional structures of members of families B (Rouvinen et al, 1990), E (Juy et al, 1992), G (Okada, 1989) and K (Davies et al, 1993) have been determined and have been shown to be widely different, suggesting that each family could have a different three-dimensional fold. Crystallizations of P-glycanases from family A (Cutfield et al, 1992), family C (Davies et al, 1992) and family F (Bedarkar et al, 1992;Pickersgill et al, 1993) have been reported and should soon provide information on whether this is the case.…”

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confidence: 99%

Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases

Schou

Rasmussen

Kaltoft

et al. 1993

European Journal of Biochemistry

101

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The catalytic activity of nine enzymes (endoglucanases 1-111, V, VI and cellobiohydrolases I and I1 from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A-C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates. For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H . insotens as a coupled enzyme using 2,6-dichloroindophenol as the electron acceptor. These data were used to estimate the number of subsites in the enzymes. The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 'H-NMR. The enzymes belonging to families which had already been investigated (A-C), showed results in agreement with previous studies. The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining-type activity and enzymes in groups J and K had inverting-type activity. The retaining endoglucanases I and I11 displayed a high glycosyltransferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.

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“…GL1, 56,57) a plant cell wall-degrading enzyme (B. subtilis unsaturated galacturonyl hydrolase) YteR, 58) and an AGE structural homolog, YihS, (unpublished) of S. typhimurium have = -barrel structures as a basic scaffold. The = -barrel structure is also found in sugarrelated enzymes such as glucoamylase, 59) endoglucanases, 60) endo/exo-cellulase, 61) -1,2-mannosidase, 62) and maltose phosphorylase, 63) as well as polysaccharide lyases. These enzymes form the = -toroid family in the SCOP database (http://scop.berkeley.edu/data/scop.b.b.…”

Section: Structural Features Of Polysaccharide Lyasementioning

confidence: 99%