Ely Morag

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family‐III cellulose‐binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine‐stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface‐exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.

The cellulosome — A treasure-trove for biotechnology

The cellulases of many cellulolytic bacteria are organized into discrete multienzyme complexes, called cellulosomes. The multiple subunits of cellulosomes are composed of numerous functional domains, which interact with each other and with the cellulosic substrate. One of these subunits comprises a distinctive new class of noncatalytic scaffolding polypeptide, which selectively integrates the various cellulase and xylanase subunits into the cohesive complex. Intelligent application of cellulosome hybrids and chimeric constructs of cellulosomal domains should enable better use of cellulosic biomass and may offer a wide range of novel applications in research, medicine and industry.

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.

Expression, purification and subunit-binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome.

The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non‐catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose‐binding domain (CBD) and nine subunit‐binding domains, or cohesin domains. Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits. Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.

A cohesin domain from Clostridium thermocellum: the crystal structure provides new insights into cellulosome assembly.

BACKGROUND The scaffoldin component of the cellulolytic bacterium Clostridium thermocellum is a non-hydrolytic protein which organizes the hydrolytic enzymes in a large complex, called the cellulosome. Scaffoldin comprises a series of functional domains, amongst which is a single cellulose-binding domain and nine cohesin domains which are responsible for integrating the individual enzymatic subunits into the complex. The cohesin domains are highly conserved in their primary amino acid sequences. These domains interact with a complementary domain, termed the dockerin domain, one of which is located on each enzymatic subunit. The cohesin-dockerin interaction is the crucial interaction for complex formation in the cellulosome. The determination of structural information about the cohesin domain will provide insights into cellulosome assembly and activity. RESULTS We have determined the three-dimensional crystal structure of one of the cohesin domains from C. thermocellum (cohesin 2) at 2.15 A resolution. The domain forms a nine-stranded beta sandwich with a jelly-roll topology, somewhat similar to the fold displayed by its neighboring cellulose-binding domain. CONCLUSIONS The compact nature of the cohesin structure and its lack of a defined binding pocket suggests that binding between the cohesin and dockerin domains is characterized by interactions between exposed surface residues. As the cohesin-dockerin interaction appears to be rather nonselective, the binding face would presumably be characterized by surface residues which exhibit both intraspecies conservation and interspecies dissimilarity. Within the same species, unconserved surface residues may reflect the position of a given cohesin domain within the scaffoldin subunit, its orientation and interactions with neighboring domains.

Cohesin-dockerin microarray : Diverse specificities between two complementary families of interacting protein modules

The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin‐dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules. Matching recombinant fusion proteins were prepared that contained one of the interacting modules: cohesins were joined to an appropriate cellulose‐binding module (CBM) and the dockerins were fused to a thermostable xylanase that served to enhance expression and proper folding. The CBM‐fused cohesins were immobilized on cellulose‐coated glass slides, to which xylanase‐fused dockerin samples were applied. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of the parent cellulosome and allows selection of suitable cohesin‐dockein pairs for biotechnological and nanotechnological application. Using this approach, extensive cross‐species interaction among type‐II cohesins and dockerins is shown for the first time. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated.

Affinity digestion for the near-total recovery of purified cellulosome from Clostridium thermocellum

Abstract The affinity properties displayed by the cellulosome, the multienzyme complex from Clostridium thermocellum , for its insoluble polymeric substrate, cellulose, have previously been employed for its purification by affinity chromatography. In the present communication, a new purification procedure is described which provides five-fold higher yields (>90%) of the cellulosome with enhanced solubilizing activity (approximately 1.5-fold). The new method is based on the efficient adsorption of the cellulosome onto phosphoric acid-treated substrate (amorphous cellulose) and the subsequent digestion of the carrier/substrate by the adsorbed enzyme complex. The term “affinity digestion” is proposed for such systems in which the affinity matrix is degraded totally by the adsorbed enzyme(s), thus facilitating its recovery.

Regulation of the Cellulosomal celS (cel48A) Gene of Clostridium thermocellum Is Growth Rate Dependent

Clostridium thermocellum produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures. The transcriptional level of celS mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation. The amount of celS mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h(-1) and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h(-1), respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h(-1), respectively. Two major transcriptional start sites were detected at positions -140 and -145 bp, upstream of the translational start site of the celS gene. The potential promoters exhibited homology to known sigma factors (i.e., sigma(A) and sigma(B)) of Bacillus subtilis. The relative activity of the two promoters remained constant under the conditions studied and was in agreement with the results of the RNase protection assay, in which the observed transcriptional activity was inversely proportional to the growth rate.

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.

Deconstruction of Lignocellulose into Soluble Sugars by Native and Designer Cellulosomes

ABSTRACT Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes of Clostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides. IMPORTANCE Cellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacterium Thermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels. Cellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacterium Thermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels.