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Exploration of new geometries in cellulosome-like chimeras.

ABSTRACT: In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a "covalent cellulosome" that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.

SUBMITTER: Mingardon F

PROVIDER: S-EPMC2168198 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Exploration of new geometries in cellulosome-like chimeras.

Mingardon Florence F Chanal Angélique A Tardif Chantal C Bayer Edward A EA Fierobe Henri-Pierre HP

Applied and environmental microbiology 20070928 22

In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cell ...[more]

PMID: 17905885

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Project description:The chimeric sequences produced by phi29 DNA polymerase, which are named as chimeras, influence the performance of the multiple displacement amplification (MDA) and also increase the difficulty of sequence data process. Despite several articles have reported the existence of chimeric sequence, there was only one research focusing on the structure and generation mechanism of chimeras, and it was merely based on hundreds of chimeras found in the sequence data of E. coli genome.We finished data mining towards a series of Next Generation Sequencing (NGS) reads which were used for whole genome haplotype assembling in a primary study. We established a bioinformatics pipeline based on subsection alignment strategy to discover all the chimeras inside and achieve their structural visualization. Then, we artificially defined two statistical indexes (the chimeric distance and the overlap length), and their regular abundance distribution helped illustrate of the structural characteristics of the chimeras. Finally we analyzed the relationship between the chimera type and the average insertion size, so that illustrate a method to decrease the proportion of wasted data in the procedure of DNA library construction.131.4 Gb pair-end (PE) sequence data was reanalyzed for the chimeras. Totally, 40,259,438 read pairs (6.19%) with chimerism were discovered among 650,430,811 read pairs. The chimeric sequences are consisted of two or more parts which locate inconsecutively but adjacently on the chromosome. The chimeric distance between the locations of adjacent parts on the chromosome followed an approximate bimodal distribution ranging from 0 to over 5,000 nt, whose peak was at about 250 to 300 nt. The overlap length of adjacent parts followed an approximate Poisson distribution and revealed a peak at 6 nt. Moreover, unmapped chimeras, which were classified as the wasted data, could be reduced by properly increasing the length of the insertion segment size through a linear correlation analysis.This study exhibited the profile of the phi29MDA chimeras by tens of millions of chimeric sequences, and helped understand the amplification mechanism of the phi29 DNA polymerase. Our work also illustrated the importance of NGS data reanalysis, not only for the improvement of data utilization efficiency, but also for more potential genomic information.

Project description:BackgroundRenewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria.ResultsHere, we report the design and function of a novel hyperthermostable "designer cellulosome" system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum.ConclusionThe functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

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Exploration of new geometries in cellulosome-like chimeras.

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Exploration of new geometries in cellulosome-like chimeras.

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