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, Takao; Kawamoto, Noriko; Shinya, Shoko; Okazaki, Nobuo; Nakazawa, Masami; Miyatake, Kazutaka; Fukamizo, Tamo; Ueda, Mitsuhiro; Tamada, Taro

doi:10.1074/jbc.m113.462135

Cited by 39 publications

(29 citation statements)

References 47 publications

(71 reference statements)

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“…The C′D′ subsites were shifted compared to those in the crystal structure as can be seen in Figure D. A similar binding mode is seen in the complex of Ra‐ChiC, a lysozyme‐like chitinolytic enzyme, with NAG4 (PDB ID 3W6C; Figure S3) which has the highest sequence similarity among the structural representatives of λ lysozyme in the Protein Data Bank (PDB, http://www.rcsb.org ).…”

Section: Resultssupporting

confidence: 52%

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra‐ and hexa‐N‐acetylchitohexaose

et al. 2019

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The X‐ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa‐N‐acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E′F′ sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. 1HN and 15N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme‐inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme‐peptidoglycan complex.

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Section: Resultssupporting

confidence: 52%

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra‐ and hexa‐N‐acetylchitohexaose

et al. 2019

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“…Comparison of amino acid sequence of chitinases reveals two highly conserved motifs in family GH18 chitinase: D XX D X D X E and S X GG correspond to the catalytic domain and substrate-binding site, respectively (Henrissat and Bairoch 1996). Glu and Asp residues are highly conserved within the catalytic domain of chitinases indicating their direct involvement in hydrolysis of the glycosidic bond (Arimori et al 2013). Degradation of chitin begins when insoluble chitin binds to the chitin-binding domain.…”

Section: Fungal Chitinasesmentioning

confidence: 97%

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications

et al. 2015

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Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of chitinases, of which chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanuginosus and its industrial application.

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“…A GH23 protein encoded by a goose-type (G-type) lysozyme gene from a moderately thermophilic bacterium, Ralstonia sp. strain A-471, displays chitinolytic activity but not lysozyme activity (13,14). A GH48 protein (APAP I) from the leaf beetle, Gastrophysa atrocyanea, also exhibits chitinase activity (15), while most other members in this family display hydrolytic activity for cellulose or its derivatives.…”

mentioning

confidence: 99%

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confidence: 99%

A Structurally Novel Chitinase from the Chitin-Degrading Hyperthermophilic Archaeon Thermococcus chitonophagus

Horiuchi

Aslam

Kanai

et al. 2016

Appl Environ Microbiol

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A structurally novel chitinase, Tc-ChiD, was identified from the hyperthermophilic archaeon Thermococcus chitonophagus, which can grow on chitin as the sole organic carbon source. The gene encoding Tc-ChiD contains regions corresponding to a signal sequence, two chitin-binding domains, and a putative catalytic domain. This catalytic domain shows no similarity with previously characterized chitinases but resembles an uncharacterized protein found in the mesophilic anaerobic bacterium Clostridium botulinum. Two recombinant Tc-ChiD proteins were produced in Escherichia coli, one without the signal sequence [TcChiD(⌬S)] and the other corresponding only to the putative catalytic domain [Tc-ChiD(⌬BD)]. Enzyme assays using N-acetylglucosamine (GlcNAc) oligomers indicated that both proteins hydrolyze GlcNAc oligomers longer than (GlcNAc) 4 . Chitinase assays using colloidal chitin suggested that Tc-ChiD is an exo-type chitinase that releases (GlcNAc) 2 or (GlcNAc) 3 . Analysis with GlcNAc oligomers modified with p-nitrophenol suggested that Tc-ChiD recognizes the reducing end of chitin chains. While TcChiD(⌬BD) displayed a higher initial velocity than that of Tc-ChiD(⌬S), we found that the presence of the two chitin-binding domains significantly enhanced the thermostability of the catalytic domain. In T. chitonophagus, another chitinase ortholog that is similar to the Thermococcus kodakarensis chitinase ChiA is present and can degrade chitin from the nonreducing ends. Therefore, the presence of multiple chitinases in T. chitonophagus with different modes of cleavage may contribute to its unique ability to efficiently degrade chitin. IMPORTANCEA structurally novel chitinase, Tc-ChiD, was identified from Thermococcus chitonophagus, a hyperthermophilic archaeon. The protein contains a signal peptide for secretion, two chitin-binding domains, and a catalytic domain that shows no similarity with previously characterized chitinases. Tc-ChiD thus represents a new family of chitinases. Tc-ChiD is an exo-type chitinase that recognizes the reducing end of chitin chains and releases (GlcNAc) 2 or (GlcNAc) 3 . As a thermostable chitinase that recognizes the reducing end of chitin chains was not previously known, Tc-ChiD may be useful in a wide range of enzyme-based technologies to degrade and utilize chitin. Chitin is a ␤-1,4-linked insoluble linear polymer of N-acetylglucosamine (GlcNAc) and is the main component of the exoskeleton of crustaceans and insects, as well as the cell walls of fungi. Chitin is the second most abundant natural polysaccharide after cellulose, and its annual formation rate is in the order of 10 10 to 10 11 tons (1). At present, the majority of chitin remains unused, and thus the development of effective methods to convert chitin into useful biomaterials and/or bioenergy is important to maintain our supplies of edible polysaccharides, such as starch.Chitinases are enzymes responsible for the hydrolysis of chitin polymer, and they produce GlcNAc and/or its oligomers as products. Chitinases are found ...

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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

Cited by 39 publications

References 47 publications

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra‐ and hexa‐N‐acetylchitohexaose

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra‐ and hexa‐N‐acetylchitohexaose

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications

A Structurally Novel Chitinase from the Chitin-Degrading Hyperthermophilic Archaeon Thermococcus chitonophagus

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