Crystal Structure of Family GH-8 Chitosanase with Subclass II Specificity from Bacillus sp. K17

Adachi, Wataru; Sakihama, Yuri; Shimizu, Shinji; Sunami, Tomoko; Fukazawa, Tetsuya; Suzuki, M.; Yatsunami, Rie; Nakamura, Satoshi; Takénaka, Akio

doi:10.1016/j.jmb.2004.08.028

Cited by 110 publications

(100 citation statements)

References 37 publications

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“…72) Adachi et al showed a striking example of the replacement of the catalytic base by that from an alternative position in an inverting GH8 enzyme. 73) This result can be a cautionary notice for every researcher studying GH enzymes. The catalytic residues can not always be conserved within a GH family!…”

Section: Discussionmentioning

confidence: 94%

New Structural Insights on Carbohydrate-active Enzymes

Fushinobu¹,

Hidaka²,

Miyanaga³

et al. 2007

J. Appl. Glycosci.

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Polysaccharides show a huge degree of diversity owing to their variety of sugar components (aldoses, ketoses and their stereoisomers), the variety of glycosidic bonds (e.g., α 1,4 , α 1,6 , β 1,4 , β 1,3 , etc.), different degrees of polymerization, and different degrees of branching. Therefore, they can be involved in many types of biological function with distinct physicochemical characteristics. It is especially intriguing that α and β glucans, such as amylose (starch) and cellulose, are built from the same glucose units, differing only in the anomeric configurations of glycosidic bonds. Enzymes that hydrolyze these glucans, amylases and cellulases, are the two largest groups of glycoside hydrolases, and have been studied for a long time. A framework to discuss a whole variety of glycoside hydrolases with their three dimensional structural aspects has been established within the last 15 years. In the late 1980s, Henrissat et al. began classification of the amino acid sequences of enzymes, focusing on cellulases and hemicellulases. 1) At the earliest stage of their classification, the α amylase family was treated as belonging to a separate system.2) In 1991, the naming system of the families changed from alphabetical to numerical, and the system expanded to 35 "glycosyl hydrolase" families, incorporating three well known amylase families, GH13 (retaining α amylase family), GH14 (inverting β amylase family), and GH15 (inverting glucoamylase family), as well as many other enzymes.3) All enzymes cleaving, modifying, and creating glycosidic bonds, and modules binding to carbohydrates have been incorporated into the CAZy database (http: www.cazy.org CAZY ). Glycoside hydrolase (GH) families now include enzymes that hydrolyze the glycosidic bonds between two or more carbohydrates or between a carbohydrate and a non carbohydrate moiety. At present, there are 101 families in the GH class ( Fig. 1; note, five have been deleted or reassigned to other classes), and it will continue to grow.The first 3 D structure of a glycoside hydrolase to be determined was that of hen egg white lysozyme (classified into GH22) in the mid 1960s.4) Even in the early 1990s, however, only a limited number of GH enzyme structures were available. Henrissat and colleagues took amino acid sequence information as the primary basis for classification, but they also used hydrophobic cluster analysis from the earliest stage, trying to incorporate as much 3 D structure based information as possible.5) The significant advances in structural studies of GH enzymes over the last decade have indicated that these enzymes show a vast array of tertiary scaffolds, described as "a marketplace of different structures". 6)Our group has been trying to solve crystal structures of useful enzymes for use in bioindustry. Two of these were the first crystal structures within the GH42 and GH57 families, 7,8) and we then began to focus on CAZy enzymes belonging to structurally unknown families. We have since solved two more GH families, and provided the first crystal s...

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Section: Discussionmentioning

confidence: 94%

New Structural Insights on Carbohydrate-active Enzymes

Fushinobu¹,

Hidaka²,

Miyanaga³

et al. 2007

J. Appl. Glycosci.

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“…In view of the sequence homology and structural similarity among all GH8a members, our results also apply to cellulases, xylanases, and other EGs belonging to subfamily GH8a. 28 …”

Section: Discussionmentioning

confidence: 99%

“…26,27 Also, a phylogenetic study showed that the putative proton acceptor is not strictly conserved in GH8; instead its location shifts within the active site. 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.…”

Section: Introductionmentioning

confidence: 99%

“…This led to a subdivision of GH8 into three subfamilies (GH8a, GH8b, and GH8c) depending on the location of the proton acceptor. 28 Moreover, kinetic studies of GH8 members that have residues corresponding to Asp278 show significant retention of activity upon mutation of the putative catalytic base. 26,27 The crystal structure of the GH8 EG has a water molecule underneath the C1′ atom in a good position to proceed with the substitution.…”

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confidence: 99%

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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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“…K17. 14) In N174 chitosanase, two acidic amino acid residues, Glu22 and Asp40, the corresponding amino acid residues of which are conserved among family-46 chitosanases, act as active centers, as follows: Glu22 donates the proton to the glycosyl oxygen atom to split the -1,4-glycosidic linkage, and the water molecule activated by the carboxylate of Asp40 then attacks the C1 carbon of the substrate sugar residue to produce an -anomer as the enzymatic product. [15][16][17] y To whom correspondence should be addressed.…”

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confidence: 99%

Molecular Characterization of a Novel Family-46 Chitosanase fromPseudomonassp. A-01

Andõ

Saito

Arai

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.

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Crystal Structure of Family GH-8 Chitosanase with Subclass II Specificity from Bacillus sp. K17

Cited by 110 publications

References 37 publications

New Structural Insights on Carbohydrate-active Enzymes

New Structural Insights on Carbohydrate-active Enzymes

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

Molecular Characterization of a Novel Family-46 Chitosanase fromPseudomonassp. A-01

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