Dataset Information

The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan.

ABSTRACT: Background:Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. Results:In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. Conclusion:This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.

SUBMITTER: Chu Y

PROVIDER: S-EPMC6892212 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan.

Chu Yindi Y Hao Zhenzhen Z Wang Kaikai K Tu Tao T Huang Huoqing H Wang Yuan Y Bai Ying Guo YG Wang Yaru Y Luo Huiying H Yao Bin B Su Xiaoyun X

Biotechnology for biofuels 20191203

<h4>Background</h4>Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the <i>Caldicellulosiruptor bescii</i> CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. <i>Cb</i>Xyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target ...[more]

PMID: 31827607

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Project description:Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (kcat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

Project description:Background:Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX. Results:Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were downregulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, showing a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions:Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases were not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader substrate range than that represented by the glycoside hydrolase family in which the enzymes were categorized.

Dataset Information

The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan.

Publications

The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan.

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