Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction

Hamamoto, Toshiro; Foong, Frances; Shoseyov, Oded; Doi, Roy H.

doi:10.1007/bf00292718

Cited by 46 publications

(32 citation statements)

References 25 publications

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“…They were very similar except that EngD had about four-times-greaterp-nitrophenyl 1-1,4-cellobiosidase activity, indicating the presence of cellobiosidase activity as originally reported (14). The purified enzymes retained the substrate specificities of the original clones (6,13,14). The specific activity of EngD was about 80 times greater than that of the unpurified periplasmic fraction of the E. coli clone, and the specific activity of EngB2 purified protein was about 70 times greater than that of the periplasmic fraction of the pC2-engB E. coli clone (6).…”

Section: Resultssupporting

confidence: 65%

“…The homology is 75% for the entire gene sequence (13). EngD has the Pro-Thr-Thr linker region seen previously in C. fimi genes (24) (6,11,12).…”

Section: Discussionmentioning

confidence: 57%

“…EngD has the Pro-Thr-Thr linker region seen previously in C. fimi genes (24) (6,11,12). The EngD carboxylterminal region had homology to C. fimi exoglucanase and endoglucanase as well as to P. fluorescens endoglucanase and xylanase (13). These regions were shown to be CBD (7)(8)(9), and since engD has some homology to these regions, it was suggestive that EngD had affinity to cellulose as well.…”

Section: Discussionmentioning

confidence: 75%

“…It was found that the specificity of C. cellulovorans endoglucanases EngB and EngD was conferred by the NH2-terminal domain and that the COOH-terminal domains of both genes had homology to enzymes from other species of cellulolytic bacteria (6,13). Thus, it would also be interesting to compare our cloned endoglucanases with each other as well as with those of other species in terms of their characteristics and cellulose affinities.…”

mentioning

confidence: 97%

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Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Foong

Doi

1992

J Bacteriol

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By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium ceflulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl 13-1,4-cellobiosidase activity. The two proteins were very homologous (80%o) up to the Pro-Thr-Thr region which divided the protein into -NH2-and -COOH-terminals. The -COOH-region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocelum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH-region of EngD, which had homology to the cellulose-binding domains of Celulomonas fimi exo-and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.Cellulose degradation is an important part of the biological recycling of carbon. Since the initial investigation of the mechanism of cellulose degradation more than 30 years ago, much progress has been achieved, especially with aerobic fungal cellulases. The degradative process can be different for different species, especially between anaerobic and aerobic organisms. With cellulolytic organisms such as Tichoderma reesei, Neocallismastix frontalis, Pseudomonas fluorescens, Cellulomonas fimi, Fibrobacter succinogenes, and Ruminococcus flavefaciens, it is not certain whether the enzymes essential for the degradation of crystalline cellulose function individually or whether they are present on the cell or substrate surface as a complex (1,15,20). There is evidence that in Clostridium thernocellum as well as in Clostridium strain C7, the enzymes are present in a complex, termed the "cellulosome" (3, 18,19,22,23). There is evidence that the enzymes present in Clostndium cellulovorans are also present in a complex. It has been shown that the native cellulase complex of C. cellulovorans is a multiprotein complex of large molecular mass. The enzymic proteins are probably organized on a nonenzymic scaffolding protein which has been cloned and sequenced (25,27,29). The aim of our investigation was to find out whether our cloned enzymes were part of the cellulase complex.Many cellulolytic enzymes have been found to contain a catalytic domain and a binding domain (5, 8-10, 16, 31, 34). It was found that the specificity of C. cellulovorans endoglucanases EngB and EngD was conferred by the NH2-terminal domain and that the COOH-terminal domains of both genes had homology to enzymes from other species of cellulolytic bacteria (6, 13). Thus, it would also be interesting t...

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Section: Resultssupporting

confidence: 65%

“…The homology is 75% for the entire gene sequence (13). EngD has the Pro-Thr-Thr linker region seen previously in C. fimi genes (24) (6,11,12).…”

Section: Discussionmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 75%

mentioning

confidence: 97%

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Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Foong

Doi

1992

J Bacteriol

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“…Unfortunately, the current level of functional characterization of GH5_4, which includes the observation that most of the characterized members have not been tested consistently on the same panel of substrates (e.g. including xyloglucan) (19,20), means that clear delineation of polysaccharide specificity in this subfamily is not straightforward. This presents a significant difficulty for in silico analysis of (meta)genomes for functional prediction, as well as for the selection and application of specific enzymes for industrial biomass utilization.…”

mentioning

confidence: 99%

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

McGregor

Morar

Fenger

et al. 2016

Journal of Biological Chemistry

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The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixedlinkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B 1 4. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixedlinkage ␤-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.

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TheClostridium cellulovorans cellulosome: An enzyme complex with plant cell wall degrading activity

Doi

Tamaru

2001

Chem. Record

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Cellulose comprises a major portion of biomass on the earth, and the turnover of this material contributes to the CO2 cycle. Cellulases, which play a major role in the turnover of cellulosic materials, have been found either as free enzymes that work synergistically, or as an enzyme complex called the cellulosome. This review summarizes some of the general properties of cellulosomes, and more specifically, the properties of the Clostridium cellulovorans cellulosome. The C cellulovorans cellulosome is an extracellular enzyme complex with a molecular weight of about 1 x 10(6), and is comprised of at least ten subunits. The major subunit is the scaffolding protein CbpA, with a molecular weight of 189,000. This nonenzymatic subunit contains a cellulose binding domain (CBD) that binds the cellulosome to the substrate, nine conserved cohesins or enzyme binding domains, and four conserved surface layer homologous (SLH) domains. It is postulated that the SLH domains help to bind the cellulosome to the cell surface. The cellulosomal enzymes include cellulases (family 5 and 9 endoglucanases and a family 48 exoglucanase), a mannanase, a xylanase, and a pectate lyase. The cellulosome is capable of converting Arabidopsis and tobacco plant cells to protoplasts. One of the endoglucanases, EngE, contains three tandemly repeated SLHs at its N-terminus, and therefore appears capable of binding to the scaffolding protein CbpA as well as to the cell surface. Cellulosomes can attack crystalline cellulose, but the free cellulosomal enzymes can attack only soluble and amorphous celluloses. Nine genes for the cellulosome are found in a gene cluster cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN. Other cellulosomal genes such as engB, engE, and engY are not linked to the major gene cluster or to each other. By determining the structure and function of the cellulosome, we hope to increase the efficiency of the cellulosome by genetic engineering techniques.

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Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction

Cited by 46 publications

References 25 publications

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

TheClostridium cellulovorans cellulosome: An enzyme complex with plant cell wall degrading activity

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