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Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.

ABSTRACT: The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055. The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative. CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily E1 endoglucanase domain, a family J endoglucanase domain, a docking domain, and another domain of unknown function (UD-2). UD-1 has no significant similarity to UD-2. CelJ hydrolyzed carboxymethylcellulose and xylan, and xylanase activity was ascribed to the family J domain. Antiserum raised against the truncated CelJ cross-reacted with proteins contained in the cellulosome of C. thermocellum F1. These results strongly suggest that CelJ is equivalent to S2, which was identified as the largest catalytic component in the cellulosome of C. thermocellum YS. A second but incomplete ORF encoding an enzyme classified in subfamily E2 endoglucanase, was located downstream of celJ.

SUBMITTER: Ahsan MM

PROVIDER: S-EPMC178413 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.

Ahsan M M MM Kimura T T Karita S S Sakka K K Ohmiya K K

Journal of bacteriology 19961001 19

The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055. The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative. CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily ...[more]

PMID: 8824619

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Project description:The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of cbhA (V. V. Zverlov et al., J. Bacteriol. 180:3091-3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate that celK and cbhA in the genome of C. thermocellum have evolved through gene duplication and recombination of domain coding sequences. celK without a dockerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65 degrees C, respectively. It hydrolyzed p-nitrophenyl-beta-D-cellobioside with a Km and a Vmax of 1.67 microM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60 degrees C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.

Dataset Information

Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.

Publications

Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.

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