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MetaCyc Enzyme: cellulase A

Gene: celA Accession Numbers: G-12684 (MetaCyc), Cthe_0269

Synonyms: endoglucanase A, endo-1,4-β-glucanase, Cel8A

Species: Ruminiclostridium thermocellum ATCC 27405

Component of:
fully loaded scaffoldin (cellobiose, high abundance catalytic units) (summary available)
cellulosome complex (or2p anchor, 18 most abundant catalytic units in cellobiose-grown cells) (extended summary available)

Summary:
The celA gene of Ruminiclostridium thermocellum encodes one of the major endoglucanases secreted by the organism. The gene has been cloned from a cosmid library, expressed in Escherichia coli [Cornet83], and sequenced [Beguin85]. The protein is a member of glycosyl hydrolase family 8, and includes a type I dockerin domain that facilitates its binding to one of the type I cohesin domains of the CipA scaffoldin protein, enabling it to become a part of the cellulosome complex.

The celA gene was successfully expressed in Saccharomyces cerevisiae, although the enzyme produced was not secreted [Benitez89]. It was also expressed in Bacillus subtilis, from which it was purified [Yao07]. The Km value for carboxymethyl cellulose (CM-cellulose) was 41 mg/ml.

The crystal structure of CelA was determined at 1.65 A resolution [Alzari96] and at atomic 0.94 A resolution [Guerin02]. The substrate occupies six D-glucosyl-binding subsites, three on either side of the scissile glycosidic linkage, explaining why family 8 cellulases cleave cello-oligosaccharide polymers that are at least five D-glycosyl subunits long. Severe torsional strain upon the bound substrate forces a distorted conformation for the glucosyl residue bound at subsite -1, which facilitates the formation of an oxocarbenium ion intermediate and might favor the breakage of the sugar ring concomitant with catalysis [Guerin02].

Locations: extracellular space

Molecular Weight of Polypeptide: 52.594 kD (from nucleotide sequence), 43.0 kD (experimental) [Yao07 ]

Unification Links: Entrez-gene:4808552 , Pride:A3DC29 , Protein Model Portal:A3DC29 , SMR:A3DC29 , String:203119.Cthe_0269 , UniProt:A3DC29

Relationship Links: CAZy:IN-FAMILY:GH8 , InterPro:IN-FAMILY:IPR002037 , InterPro:IN-FAMILY:IPR002105 , InterPro:IN-FAMILY:IPR008928 , InterPro:IN-FAMILY:IPR012341 , InterPro:IN-FAMILY:IPR016134 , InterPro:IN-FAMILY:IPR018242 , InterPro:IN-FAMILY:IPR018247 , InterPro:IN-FAMILY:IPR019834 , PDB:Structure:1CEM , PDB:Structure:1IS9 , PDB:Structure:1KWF , Pfam:IN-FAMILY:PF00404 , Pfam:IN-FAMILY:PF01270 , Prints:IN-FAMILY:PR00735 , Prosite:IN-FAMILY:PS00018 , Prosite:IN-FAMILY:PS00448 , Prosite:IN-FAMILY:PS00812

Gene-Reaction Schematic: ?

GO Terms:

Cellular Component: GO:0005576 - extracellular region

Credits:
Created 25-Mar-2011 by Caspi R , SRI International


Enzymatic reaction of: cellulase

EC Number: 3.2.1.4

cellulose + n H2O <=> n a cellodextrin

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for cellulose: carboxymethyl cellulose [Yao07 ]

pH(opt): 6.4 [Yao07]


Subunit of: fully loaded scaffoldin (cellobiose, high abundance catalytic units)

Species: Ruminiclostridium thermocellum ATCC 27405

Subunit composition of fully loaded scaffoldin (cellobiose, high abundance catalytic units) = [Cthe_0032][CelE][CelK][Cthe_0821][XghA][CelA][XynC][XynA][XynZ][CipA]
         glycosyl hydrolase family 26 domain protein = Cthe_0032 (summary available)
         cellulase E = CelE (extended summary available)
         cellulose 1,4-β-cellobiosidase (non-reducing end) = CelK (extended summary available)
         glycosyl hydrolase family 5 domain protein = Cthe_0821 (summary available)
         endo-β-1,4-glucanase XghA = XghA (extended summary available)
         cellulase A = CelA (extended summary available)
         endo-1,4-β-xylanase XynC = XynC (extended summary available)
         endo-1,4-β-xylanase XynA = XynA (extended summary available)
         endo-1,4-β-xylanase XynZ = XynZ (extended summary available)
         CipA scaffoldin = CipA (extended summary available)

Component of: cellulosome complex (or2p anchor, 18 most abundant catalytic units in cellobiose-grown cells) (extended summary available)

Summary:
This protein complex stands for a CipA scaffoldin protein loaded with the 9 most abundant catalytic cellulosomal proteins that are expressed in cells of Ruminiclostridium thermocellum grown on β-D-cellobiose (ratio of 14-5 mol catalytic unit per CipA molecule). The most abundant catalytic unit under these conditions is endo-1,4-β-xylanase XynZ, with a ratio of 14 XynZ/CipA [Gold07].

Credits:
Created 04-Apr-2011 by Caspi R , SRI International


Subunit of: cellulosome complex (or2p anchor, 18 most abundant catalytic units in cellobiose-grown cells)

Species: Ruminiclostridium thermocellum ATCC 27405

Subunit composition of cellulosome complex (or2p anchor, 18 most abundant catalytic units in cellobiose-grown cells) = [Orf2p][(Cthe_0032)(CelE)(CelK)(Cthe_0821)(XghA)(CelA)(XynC)(XynA)(XynZ)(CipA)][(CipA)(CelB)(Cthe_2193)(XynD)(CelG)(CelR)(CelF)(CbhA)(Cthe_0405)(Cthe_1271)]
         anchoring scaffoldin Orf2p = Orf2p (extended summary available)
         fully loaded scaffoldin (cellobiose, high abundance catalytic units) = (Cthe_0032)(CelE)(CelK)(Cthe_0821)(XghA)(CelA)(XynC)(XynA)(XynZ)(CipA) (summary available)
                 glycosyl hydrolase family 26 domain protein = Cthe_0032 (summary available)
                 cellulase E = CelE (extended summary available)
                 cellulose 1,4-β-cellobiosidase (non-reducing end) = CelK (extended summary available)
                 glycosyl hydrolase family 5 domain protein = Cthe_0821 (summary available)
                 endo-β-1,4-glucanase XghA = XghA (extended summary available)
                 cellulase A = CelA (extended summary available)
                 endo-1,4-β-xylanase XynC = XynC (extended summary available)
                 endo-1,4-β-xylanase XynA = XynA (extended summary available)
                 endo-1,4-β-xylanase XynZ = XynZ (extended summary available)
                 CipA scaffoldin = CipA (extended summary available)
         fully loaded scaffoldin (cellobiose, medium abundance catalytic subunits) = (CipA)(CelB)(Cthe_2193)(XynD)(CelG)(CelR)(CelF)(CbhA)(Cthe_0405)(Cthe_1271) (summary available)
                 CipA scaffoldin = CipA (extended summary available)
                 cellulase B = CelB (extended summary available)
                 glycosyl hydrolase family 5 domain protein = Cthe_2193 (summary available)
                 endo-1,4-β-xylanase XynD = XynD (extended summary available)
                 cellulase G = CelG (extended summary available)
                 cellulase R = CelR (summary available)
                 cellulase F = CelF (extended summary available)
                 cellulose 1,4-β-cellobiosidase CbhA = CbhA (extended summary available)
                 glycosyl hydrolase family 5 domain protein = Cthe_0405 (summary available)
                 glycosyl hydrolase family 43 domain protein = Cthe_1271 (summary available)

Summary:
The strictly anerobic, thermophilic bacterium Ruminiclostridium thermocellum is the microorganism with the fastest growth rate on the recalcitrant substrate crystalline cellulose [Lynd02]. The higher efficiency of its extracellular hydrolytic machinery over that of other microorganisms is due to the formation of a huge enzyme complex, the cellulosome, which has a size of 18 nm diameter and a mass in excess of 2000 kDa [Shoham99].

The cellulosome is a modular complex. The center of the complex consists of a central non-catalytic protein known as the scaffoldin (CipA), which binds up to nine catalytic subunits [Wu88]. The attachment of each catalytic subunit is mediated by the interaction of its type I dockerin domain with one of the nine type I cohesin domains of CipA [Kruus95].

CipA is, in turn, bound to the cell surface by the interaction of its type II dockerin domain with the type II cohesin domain of one of three S-layer anchor proteins, SdbA, Orf2p, or OlpB [Bayer98]. Each of these anchor proteins contains a C-terminal S-layer homology module that mediates attachment to the bacterial cell surface, and a different number of type II cohesin modules capable of binding CipA molecules. Thus different anchoring scaffoldins form cellulosome complexes of differing sizes. The smallest one, anchoring scaffoldin SdbA, contains one cohesin domain, and thus binds one CipA molecule along with the nine catalytic units bound to it. The medium size anchoring scaffoldin Orf2p contains two cohesin domains and thus binds two CipA molecules and forms complexes with 18 catalytic units. The most abundant anchor protein is anchoring scaffoldin OlpB, which contains seven cohesin domains and forms complexes of up to 63 catalytic units.

In addition to nine type I cohesin domains and a type II dockerin domain, the CipA scaffoldin also contains a type III cellulose-binding module for attachment of the complex to cellulose [Gerngross93]. Many of the catalytic units also contain carbohydrate-binding domains with differing specificities.

The specific composition of the cellulosomes depends on growth rate and the nature of substrates available. In a 2007 experiment that identified cellulosome components under conditions of growth on either β-D-cellobiose or avicel [Gold07], the following components were detected (in order of abundance):

Proteins detected in avicel-grown cells:

cellulose 1,4-β-cellobiosidase (non-reducing end), cellulose 1,4-β-cellobiosidase (reducing end), cellulase R, anchoring scaffoldin OlpB, Cthe_0821 glycosyl hydrolase family 5 domain protein, cellulase A, cellulase E, cellulase J, endo-1,4-β-xylanase XynC, endo-1,4-β-xylanase XynZ, cellulose 1,4-β-cellobiosidase CbhA, cellulase T, cellulase G, endo-1,4-β-xylanase XynA, endo-β-1,4-glucanase XghA, cellulase W, cellulase F, Cthe_0736 cellulosomal cohesin-domain protein, Cthe_2193 glycosyl hydrolase family 5 domain protein, licheninase LicB, mannanase A, cellulase Q, Cthe_0032 glycosyl hydrolase family 26 domain protein, cellulase B, anchoring scaffoldin Orf2p, cellulase P, Cthe_2761 glycosyl hydrolase family 9 domain protein, Cthe_0405 glycosyl hydrolase family 5 domain protein, cellulase N, cellulase D, glycoside hydrolase family 9 domain protein LecB, endochitinase ChiA, Cthe_1400 glycosyl hydrolase 53 domain protein, pectin lyase.

The following proteins were detected only in avicel-grown cells:

Cthe_0736 cellulosomal cohesin-domain protein, licheninase LicB, mannanase A, cellulase Q, cellulase P, cellulase N, and pectin lyase.

Proteins detected in cellobiose-grown cells:

endo-1,4-β-xylanase XynZ, CipA scaffoldin, endo-1,4-β-xylanase XynA, endo-1,4-β-xylanase XynC, cellulase A, endo-β-1,4-glucanase XghA, Cthe_0821 glycosyl hydrolase family 5 domain protein, cellulose 1,4-β-cellobiosidase (non-reducing end), cellulase E, Cthe_0032 glycosyl hydrolase family 26 domain protein, cellulase B, anchoring scaffoldin OlpB, Cthe_2193 glycosyl hydrolase family 5 domain protein, endo-1,4-β-xylanase XynD, cellulase G, cellulase R, cellulase F, cellulose 1,4-β-cellobiosidase CbhA, Cthe_0405 glycosyl hydrolase family 5 domain protein, Cthe_1271 glycosyl hydrolase family 43 domain protein, Cthe_2761 glycosyl hydrolase family 9 domain protein, cellulase T, cellulase W, cellulose 1,4-β-cellobiosidase (reducing end), endochitinase ChiA, cellulase J, Cthe_1400 glycosyl hydrolase 53 domain protein, glycoside hydrolase family 9 domain protein LecB, α-L-arabinofuranosidase B, anchoring scaffoldin Orf2p, cellulase D, anchoring scaffoldin SdbA, α-L-arabinofuranosidase B, endo-1,4-β-xylanase XynY.

The following proteins were detected only in β-D-cellobiose-grown cells:

endo-1,4-β-xylanase XynD, Cthe_1271 glycosyl hydrolase family 43 domain protein, α-L-arabinofuranosidase B, anchoring scaffoldin SdbA, α-L-arabinofuranosidase B and endo-1,4-β-xylanase XynY

Credits:
Created 04-Apr-2011 by Caspi R , SRI International


References

Alzari96: Alzari PM, Souchon H, Dominguez R (1996). "The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum." Structure 4(3);265-75. PMID: 8805535

Bayer98: Bayer EA, Shimon LJ, Shoham Y, Lamed R (1998). "Cellulosomes-structure and ultrastructure." J Struct Biol 124(2-3);221-34. PMID: 10049808

Beguin85: Beguin P, Cornet P, Aubert JP (1985). "Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum." J Bacteriol 162(1);102-5. PMID: 3980433

Benitez89: Benitez J, Silva A, Vazquez R, Noa MD, Hollenberg CP (1989). "Secretion and glycosylation of Clostridium thermocellum endoglucanase A encoded by the celA gene in Saccharomyces cerevisiae." Yeast 5(4);299-306. PMID: 2675490

Cornet83: Cornet P, Tronik D, Millet J, Aubert J P (1983). "Cloning and expression in Escherichia coli of Clostridium thermocellum genes coding for amino acid synthesis and cellulose hydrolysis." FEMS Microbiology Letters 16(1):137-141.

Gerngross93: Gerngross UT, Romaniec MP, Kobayashi T, Huskisson NS, Demain AL (1993). "Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology." Mol Microbiol 8(2);325-34. PMID: 8316083

Gold07: Gold ND, Martin VJ (2007). "Global view of the Clostridium thermocellum cellulosome revealed by quantitative proteomic analysis." J Bacteriol 189(19);6787-95. PMID: 17644599

Guerin02: Guerin DM, Lascombe MB, Costabel M, Souchon H, Lamzin V, Beguin P, Alzari PM (2002). "Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate." J Mol Biol 316(5);1061-9. PMID: 11884144

Kruus95: Kruus K, Lua AC, Demain AL, Wu JH (1995). "The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome." Proc Natl Acad Sci U S A 92(20);9254-8. PMID: 7568112

Lynd02: Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002). "Microbial cellulose utilization: fundamentals and biotechnology." Microbiol Mol Biol Rev 66(3);506-77, table of contents. PMID: 12209002

Shoham99: Shoham Y, Lamed R, Bayer EA (1999). "The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides." Trends Microbiol 7(7);275-81. PMID: 10390637

Wu88: Wu, J.H.D., Orme-Johnson, W.H., Demain, A.L. (1988). "Two components of an extracellular protein aggregate of Clostridium thermocellum together degrade crystalline cellulose." Biochemistry 27:1703-1709.

Yao07: Yao Q, Sun T, Chen G, Liu W (2007). "Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum." Biotechnol Lett 29(8);1243-7. PMID: 17520178


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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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