Anne Belaich

Degradation of Cellulose Substrates by Cellulosome Chimeras SUBSTRATE TARGETING VERSUS PROXIMITY OF ENZYME COMPONENTS

A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Bélaı̈ch, A., Lamed, R., Shoham, Y., Bélaı̈ch, J.-P., and Bayer, E. A. (2001)J. Biol. Chem. 276, 21257–21261), based on the high affinity species-specific cohesin-dockerin interaction. Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulose-binding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. The activities of the resultant ternary complexes were assayed using different types of cellulose substrates. Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates. On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate. For certain enzyme pairs, however, one effect appeared to predominate over the other. The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites.

Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: Prediction of specificity determinants of the dockerin domain

The cross‐species specificity of the cohesin–dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin‐containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin‐containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium‐dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin–dockerin interaction seems to be species‐specific. Based on intra‐ and cross‐species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino‐acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium‐binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Proteins 29:517–527, 1997.

Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin.

In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002) J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5.

BACKGROUND Cellulases are glycosyl hydrolases--enzymes that hydrolyze glycosidic bonds. They have been widely studied using biochemical and microbiological techniques and have attracted industrial interest because of their potential in biomass conversion and in the paper and textile industries. Glycosyl hydrolases have lately been assigned to specific families on the basis of similarities in their amino acid sequences. The cellulase endoglucanase A produced by Clostridium cellulolyticum (CelCCA) belongs to family 5. RESULTS We have determined the crystal structure of the catalytic domain of CelCCA at a resolution of 2.4 A and refined it to 1.6 A. The structure was solved by the multiple isomorphous replacement method. The overall structural fold, (alpha/beta)8, belongs to the TIM barrel motif superfamily. The catalytic centre is located at the C-terminal ends of the beta strands; the aromatic residues, forming the substrate-binding site, are arranged along a long cleft on the surface of the globular enzyme. CONCLUSIONS Strictly conserved residues within family 5 are described with respect to their catalytic function. The proton donor, Glu170, and the nucleophile, Glu307, are localized on beta strands IV and VII, respectively, and are separated by 5.5 A, as expected for enzymes which retain the configuration of the substrates anomeric carbon. Structure determination of the catalytic domain of CelCCA allows a comparison with related enzymes belonging to glycosyl hydrolase families 2, 10 and 17, which also display an (alpha/beta)8 fold.

The cellulolytic system of Clostridium cellulolyticum

Recent findings on the cellulolytic system of the mesophilic Clostridium cellulolyticum are reviewed. Six cellulases and the scaffolding protein, which are, at the present time, the known components of the cellulosome have been cloned. The catalytic and structural properties of the cloned enzymes CelA, CelC, CelD and CelF are described. It was shown that the grafting of the cellulases onto the scaffolding protein was performed using the dockerin-cohesin attachment device and was strictly dependent on the integrity of both components of the complex. The amino-acid sequences of dockerin and cohesin domains of C. cellulolyticum were compared to that of C. cellulovorans and C. thermocellum. This sequence analysis shows that domains belonging to the thermophilic or the mesophilic bacteria can be placed into two well defined groups. The genetic organization of the gene cluster of C. cellulolyticum is discussed.

Cohesin-Dockerin Recognition in Cellulosome Assembly: Experiment Versus Hypothesis

The cohesin‐dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article ( Pagés et al. , Proteins 1997;29:517–527) we predicted that four amino acid residues of the ∼70‐residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site‐directed mutagenesis of the suspected residues, whereby the species‐specificity of the cohesin‐dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177.

Production of Heterologous and Chimeric Scaffoldins by Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.

Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum

The sequence of a 5633-bp EcoRI-PvuII DNA fragment from Clostridium cellulolyticum was determined. This fragment contains two complete endo-beta-1,4-glucanase-encoding genes, designated celCCC and celCCG. These two genes are flanked by two other partial open reading frames (ORF1 and celCCE) that probably encode two cellulases or related enzymes. The celCCC and celCCG genes appear to be present in a polycistronic transcriptional unit. Northern blot hybridisations with intragenic probes derived from celCCC and celCCG gave similar patterns. Two transcripts of about 5 and 6 kb were identified. The celCCC and celCCG ORFs extend over 1380 bp and 2175 bp, respectively. They are separated by only 87 nt. A typical signal sequence is present at the N terminus of the deduced polypeptides. The mature CelCCC and CelCCG proteins have M(r)s 47,201 and 76,101, respectively. Comparisons between their amino acid (aa) sequences and other known cellulase sequences revealed that: first, they both contain the repeated 24-aa sequence characteristic of clostridial beta-glycanases, secondly, the N-terminal catalytic domains of CelCCC and CelCCG can be classified into the D and E2 families, respectively, and thirdly, the largest CelCCG contains an additional internal domain which is very similar to that of the Bacillus-type cellulose-binding domain (CBD). The ORF1-C-terminal-encoded sequence also contains the clostridial 24-aa repeat. The CelCCE N-terminus consists of a typical signal sequence followed by a 168-aa domain homologous to the N-terminal repeated domain of Cellulomonas fimi CenC. This domain is connected to an incomplete catalytic domain of family E1 by a Pro-rich junction linker.

CelE, a Multidomain Cellulase from Clostridium cellulolyticum: a Key Enzyme in the Cellulosome?

CelE, one of the three major proteins of the cellulosome of Clostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on p-nitrophenyl-cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.

Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824.

ABSTRACT The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter Pthl. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.