Molecular and Biochemical Analyses of CbCel9A/Cel48A, a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose

Yi, Zhuolin; Su, Xiaoyun; Revindran, Vanessa; Mackie, Roderick I.; Cann, Isaac

doi:10.1371/journal.pone.0084172

Cited by 67 publications

(86 citation statements)

References 52 publications

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“…This specific activity of CbXyn10C is, however, very similar and comparable to that of GH9/CBM3c module of CelA recombinantly expressed in E. coli (6). As has been observed by Zverlov et al, CelA has posttranslational modifications (22) which were regarded to be responsible for the observed difference in the specific activities between natural and recombinant CelA proteins and its truncation mutants (6). The substrate competition experiment suggested that CbXyn-10C most likely harbors only one catalytic center.…”

Section: Discussionsupporting

confidence: 73%

“…1E), as determined using beech wood xylan as the substrate. The thermophilic character of CbXyn10C is similar to that of other functionally characterized C. bescii glycoside hydrolases (4,6,22). Since C. bescii is a hyperthermophilic bacterium, this is not unexpected.…”

Section: Resultssupporting

confidence: 57%

“…Notably, however, the three multimodular enzymes differ much in their N termini: CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) has a GH9/CBM3c catalytic binary partner, C. bescii Xyn10C (CbXyn10C)/Cel48B (GenBank accession number ACM60945) has a GH10 catalytic domain, and the third protein (GenBank accession number ACM60948) has a GH74 domain. While the GH9 and GH48 catalytic domains in CelA have been extensively characterized (6,7), no analysis of the GH10 and GH74 catalytic domains has been carried out.…”

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The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

Xue

Wang

et al. 2015

Appl Environ Microbiol

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bThe genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical Cterminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley ␤-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/␤-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages. P lant cell wall polysaccharides (PCWPs), composed mainly of cellulose and hemicellulose, are a promising rich resource for renewable biofuel development (1). The complete deconstruction of PCWPs into fermentable, simple mono-or oligosaccharides requires the concerted action of a complex array of glycoside hydrolases (GHs), including cellulases and hemicellulases (2, 3). The genomes of Gram-positive bacteria of the genus Caldicellulosiruptor encode an arsenal of thermophilic plant cell wall polysaccharide-degrading enzymes (3-5), which are appealing candidates in the design of novel, robust enzyme cocktails for PCWP deconstruction.In the genome of Caldicellulosiruptor bescii, there is a gene cluster containing three genes which encode proteins harboring two tandemly linked CBM3b modules and a GH family 48 (GH48) cellobiohydrolase in the C terminus. These CBM3b and GH48 modules, as well as their linker sequences, are extremely similar at the level of the amino acid sequence (see Fig. S1 in the supplemental material). Notably, however, the three multimodular enzymes differ much in their N termini: CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) ha...

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Section: Discussionsupporting

confidence: 73%

Section: Resultssupporting

confidence: 57%

mentioning

confidence: 99%

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The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

Xue

Wang

et al. 2015

Appl Environ Microbiol

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“…Understanding the novel lignocellulolytic enzymes within this genus is important for achieving higher levels of fermentable saccharides (13). The results seen with a unique multimodular glycoside hydrolase, CelA, which functions as a bifunctional exo-and endo-glucanase, are comparable to or better than those seen with current commercial cellulase formulations (30,31). However, branched hemicelluloses, such as arabino-xylan, restrict the complete enzymatic hydrolysis of lignocellulosic biomasses.…”

Section: Discussionmentioning

confidence: 99%

Two Distinct α- l -Arabinofuranosidases in Caldicellulosiruptor Species Drive Degradation of Arabinose-Based Polysaccharides

Saleh

Han

Lü

et al. 2017

Appl Environ Microbiol

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Species in the extremely thermophilic genus Caldicellulosiruptor can degrade unpretreated plant biomass through the action of multimodular glycoside hydrolases. To date, most focus with these bacteria has been on hydrolysis of glucans and xylans, while the biodegradation mechanism for arabinose-based polysaccharides remains unclear. Here, putative ␣-L-arabinofuranosidases (AbFs) were identified in Caldicellulosiruptor species by homology to less-thermophilic versions of these enzymes. From this screen, an extracellular XynF was determined to be a key factor in hydrolyzing ␣-1,2-, ␣-1,3-, and ␣-1,5-L-arabinofuranosyl residues of arabinosebased polysaccharides. Combined with a GH11 xylanase (XynA), XynF increased arabinoxylan hydrolysis more than 6-fold compared to the level seen with XynA alone, likely the result of XynF removing arabinofuranosyl side chains to generate linear xylans that were readily degraded. A second AbF, the intracellular AbF51, preferentially cleaved the ␣-1,5-L-arabinofuranosyl glycoside bonds within sugar beet arabinan. ␤-Xylosidases, such as GH39 Xyl39B, facilitated the hydrolysis of arabinofuranosyl residues at the nonreducing terminus of the arabinosebranched xylo-oligosaccharides by AbF51. These results demonstrate the separate but complementary contributions of extracellular XynF and cytosolic AbF51 in processing the bioconversion of arabinose-containing oligosaccharides to fermentable monosaccharides.IMPORTANCE Degradation of hemicellulose, due to its complex chemical structure, presents a major challenge during bioconversion of lignocellulosic biomass to biobased fuels and chemicals. Degradation of arabinose-containing polysaccharides, in particular, can be a key bottleneck in this process. Among Caldicellulosiruptor species, the multimodular arabinofuranosidase XynF is present in only selected members of this genus. This enzyme exhibited high hydrolysis activity, broad specificity, and strong synergism with other hemicellulases acting on arabino-polysaccharides. An intracellular arabinofuranosidase, AbF51, occurs in all Caldicellulosiruptor species and, in conjunction with xylosidases, processes the bioconversion of arabinose-branched oligosaccharides to fermentable monosaccharides. Taken together, the data suggest that plant biomass degradation in Caldicellulosiruptor species involves extracellular XynF that acts synergistically with other hemicellulases to digest arabino-polysaccharides that are subsequently transported and degraded further by intracellular AbF51 to produce shortchain arabino sugars.

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“…The major enzymes are composed of multidomain structures, two GHs, and one to three CBMs (13,14,16,17). Several multidomain enzymes have been biochemically characterized (18)(19)(20)(21)(22)(23). Nevertheless, the function and biochemical properties of noncatalytic proteins secreted by Caldicellulosiruptor species are unknown.…”

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Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

et al. 2014

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Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K a ) values of 5.2 ؋ 10 6 to 44 ؋ 10 6 M ؊1 among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.

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Molecular and Biochemical Analyses of CbCel9A/Cel48A, a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose

Cited by 67 publications

References 52 publications

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

Two Distinct α- l -Arabinofuranosidases in Caldicellulosiruptor Species Drive Degradation of Arabinose-Based Polysaccharides

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

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