Tertiary Structure and Carbohydrate Recognition by the Chitin-Binding Domain of a Hyperthermophilic Chitinase from Pyrococcus furiosus

Nakamura, Tsutomu; Mine, Shouhei; Hagihara, Yoshihisa; Ishikawa, Kazuhiko; Ikegami, Takahisa; Uegaki, Koichi

doi:10.1016/j.jmb.2008.06.006

Cited by 60 publications

(69 citation statements)

References 32 publications

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“…Based on the conserved structures and glycan-binding sites in both SusD and SGBP-A, we predict that the CusD glycan-binding sites are located in similar positions. The most striking feature of the putative substrate-binding regions of both CusD I and CusD II is the presence of two Trp residues that would provide a flat platform for binding ChiOs as well as insoluble chitin, as has been observed in chitin-binding carbohydrate-binding modules (CBMs) [20, 21]. CusD I features W280 and W65, whereas CusD II displays W330 and W74 that are located at non-equivalent positions but may nevertheless have similar roles (Fig.…”

Section: Resultsmentioning

confidence: 99%

A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin

Larsbrink

Zhu

Kharade

et al. 2016

Biotechnol Biofuels

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BackgroundChitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown.ResultsHere we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm.ConclusionsBy combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0674-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin

Larsbrink

Zhu

Kharade

et al. 2016

Biotechnol Biofuels

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“…S5 in the supplemental material). According to three-dimensional (3D) structural analyses of ChBDs from TkChiA and Pf-ChiB, the chitin-binding surface is composed of three solvent-exposed tryptophan residues ( (27)(28)(29). These tryptophan residues are also conserved in both ChBDs of Tc-ChiD.…”

Section: Draft Genome Analysis Of T Chitonophagus and Identification Ofmentioning

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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“…Residues at 162 exposed sites on the primarily α-helical T4 lysozyme (PDBid 2LZM) and β-strand chitinase (PDBid 2CWR) [37] proteins were computationally mutated to create 162 single spin labeled mutants. Each of these mutants was subjected to 500 independent Rosetta relaxation trajectories in order to obtain an ensemble of allowable spin label conformations at each site.…”

Section: Resultsmentioning

confidence: 99%

RosettaEPR: Rotamer Library for Spin Label Structure and Dynamics

et al. 2013

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An increasingly used parameter in structural biology is the measurement of distances between spin labels bound to a protein. One limitation to these measurements is the unknown position of the spin label relative to the protein backbone. To overcome this drawback, we introduce a rotamer library of the methanethiosulfonate spin label (MTSSL) into the protein modeling program Rosetta. Spin label rotamers were derived from conformations observed in crystal structures of spin labeled T4 lysozyme and previously published molecular dynamics simulations. Rosetta’s ability to accurately recover spin label conformations and EPR measured distance distributions was evaluated against 19 experimentally determined MTSSL labeled structures of T4 lysozyme and the membrane protein LeuT and 73 distance distributions from T4 lysozyme and the membrane protein MsbA. For a site in the core of T4 lysozyme, the correct spin label conformation (Χ1 and Χ2) is recovered in 99.8% of trials. In surface positions 53% of the trajectories agree with crystallized conformations in Χ1 and Χ2. This level of recovery is on par with Rosetta performance for the 20 natural amino acids. In addition, Rosetta predicts the distance between two spin labels with a mean error of 4.4 Å. The width of the experimental distance distribution, which reflects the flexibility of the two spin labels, is predicted with a mean error of 1.3 Å. RosettaEPR makes full-atom spin label modeling available to a wide scientific community in conjunction with the powerful suite of modeling methods within Rosetta.

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Tertiary Structure and Carbohydrate Recognition by the Chitin-Binding Domain of a Hyperthermophilic Chitinase from Pyrococcus furiosus

Cited by 60 publications

References 32 publications

A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin

A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin

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

RosettaEPR: Rotamer Library for Spin Label Structure and Dynamics

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