Cohesin-dockerin Interactions within and between Clostridium josui and Clostridium thermocellum
Faculty of Bioresources, Mie University, Tu, Mie, Japan Journal of Biological Chemistry
(Impact Factor: 4.57).
04/2004; 279(11):9867-74. DOI: 10.1074/jbc.M308673200
The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.
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- "The dockerin module of C. thermocellum Xyn11A is another exception to the species specificity usually observed between C. thermocellum and C. josui. Jindou et al. (2004) showed that the Xyn11A dockerin has "
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ABSTRACT: Clostridial cellulosomes are cellulolytic complexes that are formed by highly specific interactions between one of the repeated cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Although Clostridium thermocellum Xyn11A dockerin has a typical C. thermocellum dockerin sequence, in which two amino acid residues are species specifically conserved within the two segments of the dockerin modules, it can recognize Clostridium josui cohesin modules in a non-species-specific manner. The importance of these two conserved amino acids, which are part of the recognition site of the cohesin and dockerin interaction, was investigated by introducing mutations into the first and/or the second segments of the Xyn11A dockerin. Mutations in the first segment did not affect the interactions between dockerin and C. thermocellum and C. josui cohesins. However, mutations in the second segment prevented binding to cohesin proteins. A second round of mutations within the first segment re-established the affinity for both the C. thermocellum and the C. josui cohesins. However, this was not observed for a 'conventional' dockerin, Xyn10C. These results suggest that the combination of the first and second dockerin segments is important for the target recognition.
Available from: uthscsa.edu
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ABSTRACT: The year 2004 represents a milestone for the biosensor research community: in this year, over 1000 articles were published describing experiments performed using commercially available systems. The 1038 papers we found represent an approximately 10% increase over the past year and demonstrate that the implementation of biosensors continues to expand at a healthy pace. We evaluated the data presented in each paper and compiled a 'top 10' list. These 10 articles, which we recommend every biosensor user reads, describe well-performed kinetic, equilibrium and qualitative/screening studies, provide comparisons between binding parameters obtained from different biosensor users, as well as from biosensor- and solution-based interaction analyses, and summarize the cutting-edge applications of the technology. We also re-iterate some of the experimental pitfalls that lead to sub-optimal data and over-interpreted results. We are hopeful that the biosensor community, by applying the hints we outline, will obtain data on a par with that presented in the 10 spotlighted articles. This will ensure that the scientific community at large can be confident in the data we report from optical biosensors.
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ABSTRACT: Biomass is originally photosynthesized from inorgainic compounds such as CO2, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant
biomass such as cellulosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized
wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions.
In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to
study their related enzymes and genes. The anaerobic and cellulolytic bacteria,Clostridium thermocellum, Clostridium stercorarium, andClostridium josui, were isolated from compost and the chitinolyticClostridium paraputrificum from beach soil andRuminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed
inEscherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex,i.e., cellulosome which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core
protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced byE. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin
with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were
combined to one gene and expressed inE. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the
dockerin-NADH dehydrogenase are planning to bind for catalyzing CO2 reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house
gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase fromC. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice
leaves in goat rumen was slightly accelerated.C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol H2/mol glucose) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene inC. paraputrficum using a modified vector ofClostridium perfringens. The hydrogen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel (RDF), were
also found to be effective in converting biomass waste to hydrogen gas.
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