Dataset Information

Molecular cloning and transcriptional and expression analysis of engO, encoding a new noncellulosomal family 9 enzyme, from Clostridium cellulovorans.

ABSTRACT: Clostridium cellulovorans produces a major noncellulosomal family 9 endoglucanase EngO. A genomic DNA fragment (40 kb) containing engO and neighboring genes was cloned. The nucleotide sequence contained reading frames for endoglucanase EngO, a putative response regulator, and a putative sensor histidine kinase protein. The engO gene consists of 2,172 bp and encodes a protein of 724 amino acids with a molecular weight of 79,474. Northern hybridizations revealed that the engO gene is transcribed as a monocistronic 2.6-kb mRNA. 5' RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) PCR analysis indicated that the single transcriptional start site of engO was located 264 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the engO promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the sigma(A) consensus promoter sequences of gram-positive bacteria. EngO contains a typical N-terminal signal peptide of 28 amino acid residues, followed by a 149-amino-acid sequence which is homologous to the family 4-9 carbohydrate-binding domain. Downstream of this domain was an immunoglobulin-like domain of 89 amino acids. The C terminus contains a family 9 catalytic domain of glycosyl hydrolase. Mass spectrometry analysis of EngO was in agreement with that deduced from the nucleotide sequence. Expression of engO mRNA increased from early to middle exponential phase and decreased during the early stationary phase. EngO was highly active toward carboxymethyl cellulose but showed no activity towards xylan. It was optimally active at 40 to 50 degrees C and pH 5 to 6. The analysis of the products from the cellulose hydrolysis through thin-layer chromatography indicated its endoglucanase activity.

SUBMITTER: Han SO

PROVIDER: S-EPMC1169505 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Molecular cloning and transcriptional and expression analysis of engO, encoding a new noncellulosomal family 9 enzyme, from Clostridium cellulovorans.

Han Sung Ok SO Yukawa Hideaki H Inui Masayuki M Doi Roy H RH

Journal of bacteriology 20050701 14

Clostridium cellulovorans produces a major noncellulosomal family 9 endoglucanase EngO. A genomic DNA fragment (40 kb) containing engO and neighboring genes was cloned. The nucleotide sequence contained reading frames for endoglucanase EngO, a putative response regulator, and a putative sensor histidine kinase protein. The engO gene consists of 2,172 bp and encodes a protein of 724 amino acids with a molecular weight of 79,474. Northern hybridizations revealed that the engO gene is transcribed a ...[more]

PMID: 15995203

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Project description:Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.

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Dataset Information

Molecular cloning and transcriptional and expression analysis of engO, encoding a new noncellulosomal family 9 enzyme, from Clostridium cellulovorans.

Publications

Molecular cloning and transcriptional and expression analysis of engO, encoding a new noncellulosomal family 9 enzyme, from Clostridium cellulovorans.

Similar Datasets

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