Cellulose utilization by
            <i>Clostridium thermocellum</i>
            : Bioenergetics and hydrolysis product assimilation

Zhang, Y.‐H. Percival; Lynd, Lee R.

doi:10.1073/pnas.0408734102

Cited by 220 publications

(184 citation statements)

References 42 publications

(44 reference statements)

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“…Synergy experiments have identified two cellulosomal enzymes, an exo-acting GH48 cellobiohydrolase that acts from the reducing end of cellulose chains and the cellotetraose-producing endo-processive GH9 endoglucanase, Cel9R (8), as central components of the C. thermocellum cellulase system (9). It has been suggested that, by generating cellotetraose as the major product from cellulose, likely through the action of Cel9R, C. thermocellum minimizes the utilization of ATP during import of glucose units (10). The cellulase activity obtained by combining cellulosomal enzymes in vitro, however, is considerably lower than that displayed by the cellulosome presented on the surface of C. thermocellum.…”

mentioning

confidence: 77%

Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

Brás¹,

Cartmell

Carvalho

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall–degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated Ct Cel124. The protein was shown to be an endo -acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo -cellulase. The crystal structure of Ct Cel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose.

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mentioning

confidence: 77%

Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

Brás¹,

Cartmell

Carvalho

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Since the discovery of the first GP, glycogen phosphorylase, in 1938 14 only 29 distinct new GP activities have been identified; the majority in the past 15 years 15 . However, given the vast number of GHs known, it seems likely that 30 is a significant underestimate of the actual number of GP activities present in nature, especially since the use of phosphorolysis to metabolize glycans is inherently more energetically favourable for a cell than hydrolysis since the released sugar-1-phosphates feed directly into the glycolysis pathway without the need for expenditure of ATP 16,17 . Given this metabolic efficiency advantage, and considering the wide range of diverse glycans that are metabolized by microbes, it is probable that glycoside phosphorylases are more widespread than previously thought.…”

Section: Introductionmentioning

confidence: 99%

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Macdonald

Patel

Larmour

et al. 2018

Journal of Biological Chemistry

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Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-D-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenol from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to Nacetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. GH3 β-glycoside phosphorylase from a metagenomic library 2 Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.Carbohydrate active enzymes (CAZymes) are the biocatalysts responsible for the assembly, degradation and modification of glycans in biological systems 1 . They are also widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel and bioproduct development [2][3][4][5] . While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive starting materials, such as nucleotide phosphosugars 6 . The high-cost of these materials makes de novo industrial-scale glycan synthesis difficult and usually non-viable.One class of CAZyme that offers a potential solution to the high costs typically associated with enzymatic glycan synthesis is that of the glycoside phosphorylases (GPs), which are increasingly being recognized and used for the biocatalysis and biotransformation of glycans 7-9 . These enzymes ordinarily carry out phosphorolysis by transferring a glycosyl moiety from the nonreducing end of a di-or polysaccharide substrate onto inorganic phosphate, thereby generating a sugar-1-phosphate 10 ( Figure 1A). GPs distinguish themselves from most CAZymes in that the hydrolytic free energy associated with the glycosidic e...

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“…According to Li et al (2011) after releasing cellulose from LCC sheath, the first step of the fermentation process is depolymerisation throughout cellulolytic bacteria, which produce cellulase. Subsequently, the cellulose disintegration products (such as cellobiose and soluble cellulodextrin of higher order) can be transformed into methane and carbon dioxide after a series of transformations (Bhadra et al 1986;Lynd et al 2002;Zhang and Lynd 2005;Jeihanipour et al 2010Jeihanipour et al , 2011.…”

Section: Introductionmentioning

confidence: 99%

Transformation of Miscanthus and Sorghum cellulose during methane fermentation

et al. 2018

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The purpose of the paper is designation of the changes in the structure of cellulose after the methane fermentation process of Miscanthus and Sorghum harvested during the growing season and afterwards. The percentage and structure of cellulose before and after fermentation were studied. Investigations into changes of the cellulose structure were conducted by the SEC, FT-IR and XRD methods. The average percentage of cellulose after the growing season for Miscanthus varieties was higher and for Sorghum varieties was lower. As a result of the fermentation, the percentage of cellulose for both investigated species harvested in two growth seasons was lower. The degree of polymerisation for the plants harvested after the growing season was lower for the most feedstock. As a result of the fermentation process, the degree of polymerization increased for each of the investigated feedstock. However, crystallinity of cellulose remained at the same level for Miscanthus and decreased for Sorghum. It was shown that changes were different in the cellulose structure of the compared species.

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Cellulose utilization by Clostridium thermocellum : Bioenergetics and hydrolysis product assimilation

Cited by 220 publications

References 42 publications

Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

Transformation of Miscanthus and Sorghum cellulose during methane fermentation

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