Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-�-glucanase Avicelase II

Bronnenmeier, Karin; Kundt, Kerstin; Riedel, Katharina; Schwarz, Wolfgang H.; Staudenbauer, Walter L.

doi:10.1099/00221287-143-3-891

Cited by 31 publications

(20 citation statements)

References 31 publications

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“…The introduction of CBM3a into either Cel48F or Cel9G improved their activity on Avicel at the apparent expense of their activity on more tractable substrates, such as PAS-cellulose. Indeed, GH48 and GH9-CBM3c en- zymes appended with an efficient CBM3 (generally CBM3b) have been discovered in various bacteria (4,15,28,31). Acetivibrio cellulolyticus was also found to produce an enzymecontaining scaffolding protein that exhibits one GH9 module (not associated with a CBM3c), one CBM3b, and seven cohesin modules (8).…”

Section: Discussionmentioning

confidence: 99%

Exploration of New Geometries in Cellulosome-Like Chimeras

Mingardon

Chanal

Tardif

et al. 2007

Appl Environ Microbiol

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In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a "covalent cellulosome" that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.

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Section: Discussionmentioning

confidence: 99%

Exploration of New Geometries in Cellulosome-Like Chimeras

Mingardon

Chanal

Tardif

et al. 2007

Appl Environ Microbiol

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“…CelF and CelS consist of a 70-kDa catalytic domain connected to their organisms' respective dockerin domains, whereas CelY is a noncellulosomal 103-kDa protein with its N-terminal catalytic domain attached via a 10-kDa domain of unknown function (DUF) to a 17-kDa cellulose-binding domain (CBM3) [18]. Thus, CelY can directly bind cellulose, whereas CelF and CelS bind their respective scaffoldins.…”

Section: Cel48 Parental Enzymesmentioning

confidence: 99%

“…Three extensively characterized Cel48 cellulases were chosen as parents for construction of the SCHEMA recombination library: CelF [10] from the mesophile Clostridium cellulolyticum ATCC 35319, CelY [18] from the thermophile Clostridium stercorarium, and CelS (also known as CelA) [19] from the thermophile Clostridium thermocellum ATCC 27405.…”

Section: Cel48 Parental Enzymesmentioning

confidence: 99%

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

et al. 2012

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Sequence diversity within a family of functional enzymes provides a platform for elucidating structure-function relationships and for protein engineering to improve properties important for applications. Access to nature's vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) -Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS -to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence-property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure-guided recombination can play in helping to identify sequencefunction relationships within a family of enzymes by supplementing natural diversity with synthetic diversity.

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“…The family IIIb CBD ORF1 exhibits only moderate sequence similarity to the partial ORF encoding a CBD directly upstream of Bacillus lautus CelA (level of sequence identity, 59%) (21), the internal CBD of Caldicellulosiruptor sp. strain Rt69B.1 multidomain XynC (level of sequence identity, 56%) (32), and the C-terminal CBD of Clostridium stercorarium CelY (level of sequence identity, 53%) (9).…”

Section: Resultsmentioning

confidence: 99%

A Gene Encoding a Novel Multidomain β-1,4-Mannanase from Caldibacillus cellulovorans and Action of the Recombinant Enzyme on Kraft Pulp

Sunna

Gibbs

Chin

et al. 2000

Appl Environ Microbiol

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Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a ␤-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85°C and pH 6.0 and was extremely thermostable at 70°C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto-and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable ␤-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.The use of hemicellulases in the manufacture of kraft pulp has been shown to promote pulp bleaching, and in most application studies the workers have focused on using xylanases (24,47,48). Using mannanases was not studied until recently, when it was shown that mannanases, acting in combination with xylanases, enhance enzyme-aided bleaching of pulps in modified kraft processes (10, 40).1,4-␤-Mannans, which are some of the major constituents of hemicellulose, are hydrolyzed to mannose by endo-acting ␤-mannanases and exo-acting ␤-mannosidases. However, due to the complexity of the polysaccharide and the presence of side chain sugars, additional enzymes, including ␣-galactosidase, ␤-glucosidase, and acetylmannan esterase, are required to completely hydrolyze mannans (34). Extremely thermophilic bacteria and hyperthermophilic archaea are known to produce several types of thermostable glycosyl hydrolases (38). Few thermostable ␤-mannanases from thermophilic bacteria have been characterized and sequenced (14,(17)(18)(19)33), while no archaeal ␤-mannanase has been described so far. The mannanases that have been sequenced belong to either glycosyl hydrolase family 5 or glycosyl hydrolase family 26 (22).Huang et al. (X. P. Huang, J. A. Hudson, F. A. Rainey, P. D. Nichols, and H. W. Morgan, submitted for publication) described a new thermophilic, spore-forming, aerobic bacterium related to the bacilli that was able to grow on crystalline cellulose, and they named this organism Caldibacillus cellulovorans. Their isolate was related to members of the genus Al...

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Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-�-glucanase Avicelase II

Cited by 31 publications

References 31 publications

Exploration of New Geometries in Cellulosome-Like Chimeras

Exploration of New Geometries in Cellulosome-Like Chimeras

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

A Gene Encoding a Novel Multidomain β-1,4-Mannanase from Caldibacillus cellulovorans and Action of the Recombinant Enzyme on Kraft Pulp

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