Cellulose, cellulases and cellulosomes.

The structural complexity and rigidity of cellulosic substrates have given rise to a phenomenal diversity of degradative enzymes--the cellulases. Cellulolytic microorganisms produce a wide variety of different catalytic and noncatalytic enzyme modules, which form the cellulases and act synergistically on their substrate. In some microbes, several types of cellulases are organized into an elaborate multifunctional supramolecular complex, known as the cellulosome. A combination of molecular genetic, biochemical, chemical, crystallographic and microscopic techniques are paving the way for new insights into both the structure of cellulose and the mechanisms of its hydrolysis.

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.

The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides.

The cellulosome is an extracellular supramolecular machine that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides. The cellulosome arrangement can also promote adhesion to the insoluble substrate, thus providing individual microbial cells with a direct competitive advantage in the utilization of the soluble hydrolysis products.

Dynamic affinity chromatography of heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative have been chromatographed on immobilized ATP, ADP and adenosine 5'-(geta, gamma-imino) triphosphate affinity chromatography columns, in the presence and in the absence of Ng-2+ or Ca-2+.ma-32-P] ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5' (beta, gamma-imino) triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the Pi-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.

Thermostable, ammonium-activated malic enzyme of Clostridium thermocellum.

"Malic" enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) was purified from Clostridium thermocellum by DEAE-cellulose, agarose-NADP and Sephadex G-200 column chromatography. The 117-fold purified "malic" enzyme displayed a maximum activity of 135 units/mg at 40 degrees C and represented 0.8% of the total cell protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of the protein suggested 90% purity and an approximate tetrameric subunit molecular weight of 40 000. The enzyme absolutely required both bivalent and monovalent cations for catalysis. Mn2+ and NH4+ were the most effective cationic activators examined. Increasing NH4+ concentration increased both enzyme activity and affinity toward L-malate. The apparent Km for L-malate was 3 X 10(-4) M at 0.4 mM NH4Cl. Enzyme activity increased linearly when temperature was raised between 22-60 degrees C and a Q10 of 2.1 was calculated from an Arrhenius plot. The enzyme was stable at heating at 60 degrees C but was denatured at higher temperatures. The enzyme half-life was 10 min at 72 degrees C. The enzyme displayed a broad pH optimum (7.2-87.2 for Tris-HCl buffer) but was inactivated by p-chloromercuribenzoate. The high thermal stability, low apparent molecular weight and NH4+ activation are properties not common to all previously described "malic" enzymes.

Comparative studies on amino and thiol groups in myosins from different sources.

Myosins from rabbit white and red skeletal, rabbit heart, fish skeletal and chicken gizzard muscles, as well as from human platelets were subjected to trinitrophenylation by trinitrobenzene sulfonate and alkylation by N-ethylmeleimide which affected their amino and thiol groups, respectively. The blocking of amino groups was carried out in the presence or in the absence of Mg-ADP and was followed both spectrophotometrically and enzymatically. Essential amino groups, whose modification throughly changes the enzymic characteristics of myosin, were found in heart and in all skeletal muscle myosins but were absent in myosins from chicken gizzard muscle and from human platelets. The reaction of these amino groups was highly retarded in the presence of Mg-ADP. Alkylation of thiols led to loss of the K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in all myosins. However, the rate of loss of activity varied from one myosin to another and, for a given myosin, was affected by the presence of nucleotides and by the value of the ionic strength. The change in Ca(2+)-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) on alkylation was influenced by the presence of Mg - ADP during the reaction. In the absence of this nucleotide, the Ca(2+)-ATPase activity increased and reached a plateau as a consequence of modification. The extent of activation largely depended on the origin of the myosin. When alkylation was carried out in the presence of Mg-ADP, the Ca(2+)-ATPase activity as a function of time exhibited a maximum but the descending part of the curve was absent in myosins from heart and gizzard muscles.

Affinity chromatography of heavy meromyosin subfragment-1 reacted with thiol reagents.

Separation of heavy meromyosin subfragment-1 treated with N-ethyl maleimide (MalNEt) into native -SH1- and -(SH1, SH2)-blocked protein populations could be achieved by affinity chromatography on agarose-ATP columns in the presence of Mg2+ or Ca2+. Covalent bridging of the two -SH groups by p-phenylenedimaleimide gave a product which has the same affinity of binding to ATP columns as the doubly blocked MalNEt preparation. Treatment with p-phenylenedimaleimide abolished binding to immobilized F-actin columns, whereas modifications by MalNEt did not affect adsorption by this chromatographic medium. Affinity chromatography on immobilized nucleotide and actin columns is suggested as an analytical tool in the study of the involvement of thiol groups in the myosin active site and its conformation.

Possible uncoupling of the mechanochemical process in the actomyosin system by covalent crosslinking of F-actin.

It has been speculated that F-actin depolymerizes during muscle contraction. In order to test this speculation, G-actin in F-actins were immobilized by crosslinking. While this did not affect activation of myosin ATPase, superprecipitation could be abolished. Sedimentation, light scattering, electron microscope, electrophoresis and homodyne spectra measurements seem to indicate that the average size of crosslinked segments increases with time of treatment with the crosslinking agent. At the same time, the amounts of dimers and trimers decreased. It appears that crosslinking makes F-actin filaments less flexible. The possible reasons for the loss of the capability to exhibit superprecipitation are discussed.

Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulosome from Clostridium thermocellum.

The cellulose-binding domain from the scaffoldin subunit of the cellulosome from Clostridium thermocellum strain YS has been expressed in Escherichia coli, purified to homogeneity, and crystallized. Crystals were grown by vapor diffusion using polyethylene glycol as precipitant. They belong to the monoclinic space group C2 with unit cell dimensions of a = 64.68 A, b = 50.36 A, c = 96.27 A; beta = 99.43 degrees, and density packing considerations suggest that the asymmetric unit contains two molecules. The crystals diffract beyond 2.0 A resolution using a laboratory rotating anode source.

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.

Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: overview and feasibility: properties of diazirine as a precursor.

Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra :CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.

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.

Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering.

Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vitro evolution of these biologically important molecules. However, the general usefulness of this technology is still limited by some technical difficulties. One of the most debilitating obstacles to the widespread application of the technology is the accumulation of "insert loss" clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted. Another difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned. Here, we report the construction of a novel phage-display system designed to address these problems. In our system a single-chain Fv (scFv) is expressed as an in-frame fusion protein with a cellulose-binding domain (CBD) derived from the Clostridium thermocellum cellulosome. The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof. We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires.

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.

Expression, purification and crystallization of a cohesin domain from the cellulosome of Clostridium thermocellum.

The cellulosome of the cellulolytic bacterium, Clostridium thermocellum, is a multi-enzyme complex in which the enzymatic (cellulolytic) subunits are attached to a unique nonhydrolytic subunit called scaffoldin. The attachment is mediated by two mutually interacting domains: namely multiple cohesin domains on the scaffoldin subunit and a dockerin domain on each of the enzymatic subunits. Knowledge of the three-dimensional structure of each of the interacting components would be critical to a better understanding of the cohesin-dockerin interaction at the molecular level. In this report, we describe the purification of one of the nine cohesin domains of the scaffoldin subunit from C. thermocellum. A DNA segment containing the cohesin 2 sequence was fused to a hexa-histidine tag, and the resultant construct was expressed in Escherichia coli. The expressed peptide was efficiently isolated by metal-chelate affinity chromatography. The purified recombinant form of the cohesin was crystallized pending determination of its structure.

Anomalous dissociative behavior of the major glycosylated component of the cellulosome of Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is a highly cohesive multienzyme complex that is capable of completely solubilizing insoluble cellulose. One of the major cellulosomal components, the glycosylated S1 subunit, is believed to play an important structural role and normally migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 210,000. It is shown here that by simply altering the conditions (pH or ionic strength) of the environment prior to electrophoresis, a different migratory profile for S1 emerges, yielding a collection of bands, all of which migrate faster than the parent band. The original electrophoretic behavior of S1 can be reproduced on restoration of the original pH and ionic strength. These results may bear important significance for the physiological role of the S1 subunit in facilitating the observed synergistic action of the other (cellulolytic) components of the cellulosome.

Cellulase Ss (CelS) is synonymous with the major cellobiohydrolase (subunit S8) from the cellulosome of Clostridium thermocellum.

The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved. The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component Ss (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.

Nonproteolytic cleavage of aspartyl proline bonds in the cellulosomal scaffoldin subunit from Clostridium thermocellum.

Previous work from our group [Morage (Morgenstern), E., Bayer, E. A., and Lamed, R. (1991), Appl. Biochem. Biotechnol. 30, 129-136] has demonstrated an anomalous electrophoretic mobility pattern for scaffoldin, the 210-kDa cellulosome-integrating subunit of Clostridium thermocellum. Subsequent evidence [Morag, E., Bayer, E. A., and Lamed, R. (1992), Appl. Biochem. Biotechnol. 33, 205-217] indicated that the effect could be attributed to a nonproteolytic fragmentation of the subunit into a defined series of lower-molecular-weight bands. In the present work, a recombinant segment of the scaffoldin subunit was employed to determine the site(s) of bond breakage. An Asp-Pro sequence within the cohesin domain was identified to be the sensitive peptide bond. This sequence appears quite frequently in the large cellulosomal proteins, and the labile bond may be related to an as yet undescribed physiological role in the hydrolysis of cellulose by cellulosomes.