Structural and functional analyses of catalytic domain of GH10 xylanase from <i>Thermoanaerobacterium saccharolyticum</i> JW/SL‐YS485

Han, Xu; Shang, Na; Huang, Chun‐Hsiang; Ko, Tzu‐Ping; Chen, Chun‐Chi; Chan, H.C.; Cheng, Ya‐Shan; Zhu, Zhaohai; Wiegel, Juergen; Luo, Wenjun; Guo, Rey‐Ting; Ma, Yanhe

doi:10.1002/prot.24286

Cited by 14 publications

(15 citation statements)

References 26 publications

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“…2B). The -2 subsite motif in Pm25_cluster is glycine instead of glutamate in main cluster, and both glycine and glutamate at -2 subsite were found in other enyzmes (58)(59)(60). Importantly, the distance between β3 motif and β4 motif is wider in Pm25_cluster (620 amino acids) than in main cluster (92 amino acids), which indicated interrupted catalytic domains in Pm25_cluster.…”

Section: Bioinformatics Analysis Of the Pm25-encoding Gene Sequencementioning

confidence: 99%

Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome

Ioannou

Henrissat

et al. 2021

Appl Environ Microbiol

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The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan degrading enzymes including Pm25, a multi-modular Glycoside Hydrolase (GH) family 10. Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two Carbohydrate Binding Modules (CBMs) from family 4. The genes upstream of pm25 are susC-susD-unk suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization loci. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture, and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating its importance in glycan foraging for the gut microbiota. In order to understand its unusual multi-modularity and the possible role of the CBMs, a detailed characterization of the full length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific towards the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria. IMPORTANCE: Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Bacteroidetes, one of the main phyla in Rumen and Human gut microbiota, have been shown to encode polysaccharide utilization loci (PUL) dedicated to the degradation of xylan. Here we present the biochemical characterization of a xylanase encoded by a bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multi-modular enzyme of which sequence is interrupted by the insertion of two CBM from family 4. Our results show that not only this enzyme resemble homologues that were shown to be important for xylan degradation in rumen or human diet but show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilisation system. This study shed light on a better understanding toward xylan degradation and plant cell wall deconstruction which can conduct to several applications in food, feed and bioeconomy.

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Section: Bioinformatics Analysis Of the Pm25-encoding Gene Sequencementioning

confidence: 99%

Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome

Ioannou

Henrissat

et al. 2021

Appl Environ Microbiol

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“… Sequence alignment of Xyn10A with other members in the GH10 superfamily. Xyn10A: (GenBank: AEE64767.1) [ 13 ]; 3W24: intracellular xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485(PDB Id 3W24) [ 7 ]; 2Q8X: the thermophilic, intracellular xylanase from G. Stearothermophilus (PDB Id 2Q8X) [ 38 ]; 2W5F: the N -terminal endo-1,4-β- d -xylanase 10 b (Xyn10b) of C. thermocellum (PDB Id 2W5F) [ 19 ]. Strictly conserved residues are highlighted by red background and conservatively substituted residues are boxed.…”

Section: Resultsmentioning

confidence: 99%

“…In family GH10 enzymes, hydrolysis often occurs between the −1 and +1 subsites, and thus the subsite −1 essentially comprises the active site. According to the sequence to the templates Xyn10b (PDB Id 2W5F) [ 19 ] and xylanase from Thermoanaerobacterium saccharolyticum (PDB Id 3W24) [ 7 ], in subsite −1 for Xyn10A, there are seven residues: Gln263, Lys83, W336, Glu188, Glu294, His128, Met87, His265, and Trp344. All these residues are conservative in family GH10 seen from Table 3 .…”

Section: Resultsmentioning

confidence: 99%

“…The main chain of xylan is composed of 1,4- d -xylose subunits, which is usually decorated with various side chain residues of 1,2-α- d -glucuronic acid, or its 4- O -methyl ethers, 1,3-α- l -arabinose, and/or O -acetyl groups in the 2 and 3 positions. Due to structural complexity, several xylanolytic enzymes are required to release the substituents and sugars from the various xylans, including endo-1,4-β-xylanases (EC 3.2.1.8) [ 7 ], acetyl xylan esterases (EC 3.1.1.72) [ 8 ], feruloyl esterases (EC 3.1.1.73) [ 9 ], α- l -arabinofuranosidases (EC 3.2.1.55) [ 10 ], α-glucuronidases (EC 3.2.1.139) [ 11 ], and β- d -xylosidase (EC 3.2.1.37) [ 12 ]. Among these enzymes, endo-1,4-β-xylanases are believed to be the most valuable in industrial applications.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Modeling and MM-PBSA Free Energy Analysis of Endo-1,4-β-Xylanase from Ruminococcus albus 8

Zhan

Jin

et al. 2014

IJMS

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Endo-1,4-β-xylanase (EC 3.2.1.8) is the enzyme from Ruminococcus albus 8 (R. albus 8) (Xyn10A), and catalyzes the degradation of arabinoxylan, which is a major cell wall non-starch polysaccharide of cereals. The crystallographic structure of Xyn10A is still unknown. For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme. In this study, the best similarity was found with the Clostridium thermocellum (C. thermocellum) N-terminal endo-1,4-β-d-xylanase 10 b. Following the 100 ns molecular dynamics (MD) simulation, a reliable model was obtained for further studies. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods were used for the substrate xylotetraose having the reactive sugar, which was bound in the −1 subsite of Xyn10A in the 4C1 (chair) and 2SO (skew boat) ground state conformations. According to the simulations and free energy analysis, Xyn10A binds the substrate with the −1 sugar in the 2SO conformation 39.27 kcal·mol−1 tighter than the substrate with the sugar in the 4C1 conformation. According to the Xyn10A-2SO Xylotetraose (X4(sb) interaction energies, the most important subsite for the substrate binding is subsite −1. The results of this study indicate that the substrate is bound in a skew boat conformation with Xyn10A and the −1 sugar subsite proceeds from the 4C1 conformation through 2SO to the transition state. MM-PBSA free energy analysis indicates that Asn187 and Trp344 in subsite −1 may an important residue for substrate binding. Our findings provide fundamental knowledge that may contribute to further enhancement of enzyme performance through molecular engineering.

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“…TsXy1A and XynA, we infer that Glu182 and Glu280 are putative catalytic residues of XynA (25)(26)(27)(28). Of note, only the GH10 domain is in the other protein structures mentioned above, the N-terminal and C-terminal domains are unique for XynA ( Figure 2B).…”

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confidence: 86%

Structural and biochemical characterization of bifunctional XynA

Xie

Liu

et al. 2020

Preprint

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Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

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Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

Cited by 14 publications

References 26 publications

Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome

Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome

Molecular Modeling and MM-PBSA Free Energy Analysis of Endo-1,4-β-Xylanase from Ruminococcus albus 8

Structural and biochemical characterization of bifunctional XynA

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