Biomedical subjects

R Lamed

Publications and source records attributed to R Lamed.

At least 37 records · Page 2Linked to original sources

PubMed1997

Crystallization and preliminary X-ray analysis of a cohesin domain of the cellulosome from Clostridium thermocellum.

Recombinant cohesin-2, a unique type of protein-recognition domain from the cellulosome of Clostridium thermocellum, has been crystallized by the hanging-drop vapor-diffusion method. The crystals are monoclinic, space group C2 with unit-cell dimensions a = 79.91, b = 47.86, c = 51.13 A, beta = 126.77 degrees. There is most likely to be one molecule per asymmetric unit, corresponding to a packing density of 2.16 A(3) Da(-1). The crystals diffract to beyond 2.3 A on a conventional laboratory rotating-anode source.

Journal Article↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum.

The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.

Affinity Labels↗

PubMed1994

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.

Cellulase↗

PubMed1994

Flow cytometric analysis of entire microbial colonies.

Much could be gained if the scope of flow cytometry could be broadened to the study of entire cell colonies, rather than to populations of single, separate cells. This can be achieved by encapsulating single microbial cells in small spheres in a way that allows each cell to multiply and form a colony within its respective microbead, which is then amenable to analysis by flow cytometry. Methods for performing the encapsulation within beads of appropriate size and homogeneity have been developed (Nir et al., Appl. Environ. Microbiol. 56:2870-2875, 1990). We describe here how a variety of properties of the entrapped colonies, such as mass, growth rate, enzymatic activity, and expression of specific antigens, can be measured, and we discuss how these constructs can be utilized to select microbial mutants.

Animals↗

PubMed1994

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.

Bacteria↗

PubMed1993

The nature of the carbohydrate-peptide linkage region in glycoproteins from the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens.

The cellulase complexes of two cellulolytic bacteria, Clostridium thermocellum and Bacteroides cellulosolvens, were subjected to extensive Pronase digestion. Glycopeptide fractions were isolated by gel permeation and fast protein liquid chromatography and analyzed by monosaccharide analysis, amino acid analysis, methylation analysis, and 1H NMR spectroscopy. Alkaline borohydride-induced deglycosylation/amino acid conversion and periodate oxidation studies on the glycopeptide fraction of the C. thermocellum cellulosome demonstrated that the earlier established collection of carbohydrate moieties with 3-O-Me-D-GlcpNAc-alpha (1-->2)-[D-Galp-alpha (1-->3)]-D-Galf-alpha (1-->2)-D-Gal (where 3-O-Me-D-GlcpNAc is 3-O-methyl-N-acetylglucopyranosamine, Galp is galactopyranose, and Galf is galactofuranose) as the major component, is O-linked to threonine via galactopyranose. Using the same approach for the glycopeptide fraction of the cellulase complex of B. cellulosolvens, it was found that the reported collection of carbohydrate moieties with D-Galf-alpha (1-->3)-D-GlcpNAc-alpha (1-->2)-D-Galf-alpha (1-->2)-[D-Galf-beta (1-->3)]-D-Gal as the major component, is O-linked mainly to threonine and partly to serine via galactopyranose. In both species, the hydroxyamino-acid-bound galactopyranose residue has probably an alpha-configuration. The carbohydrate chains appear as clusters located in highly Thr/Pro-rich peptide regions of the glycoproteins. The results are consistent with the notion that the glycosylation sites are localized in linker sequences which connect the various binding domains of the noncatalytic S1 subunit of the cellulosome.

Bacteroides↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1992

Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.

The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens.

Amidohydrolases↗

PubMed1992

The cellulose paradox: pollutant par excellence and/or a reclaimable natural resource?

The various aspects of cellulose as a pollutant are considered in view of its lack of toxicity on the one hand and its recalcitrant durable nature on the other. The microbial degradation of cellulosics is discussed, and the contrast between its success in handling natural cellulosic wastes versus its failure to cope with man-made refuse is described. Research carried out in the past decade has demonstrated that cellulolytic organisms are provided with cell surface multifunctional multienzyme conglomerates, called cellulosomes, which are capable of solubilizing solid cellulosic substrates. The intriguing properties of such complexes include their cohesive nature, their many enzymatic components, and a characteristic glycosylated cellulose-binding, 'scaffolding' component. The latter appears to serve as a substrate-targeting carrier, which delivers the other (hydrolytic) components to the cellulose. Progress in establishing efficient model systems for in vitro solubilization of purified cellulose or natural cellulosic substrates has been achieved using purified cellulosome preparations, fortified with beta-glucosidase and pectinase. The latter enzymes were required in order to alleviate the phenomenon of product inhibition which reduces the efficiency of the free cellulosome. Such combined enzyme systems are proposed as examples of future tailor-made cellulolytic systems for the degradation of natural cellulosics.

Biodegradation, Environmental↗

PubMed1991

Primary structure of O-linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains.

The cell-free forms of the multiple cellulase-containing protein complex (cellulosome), isolated from the cellulolytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5-7% (by mass), consisting of O-linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast-atom-bombardment mass spectrometry (FAB-MS). In addition to the previously described major oligosaccharide, (formula; see text) [Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027-1035], the following partial structures of this compound could be established: (formula; see text). Cell-free and cell-associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.

Bacterial Proteins↗

PubMed1991

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.

Biotechnology↗

PubMed1991

Influence of CO(2)-HCO(3) Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens.

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO(2)-HCO(3) growth conditions and suggest that there exists a threshold level of CO(2) for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO(2)-HCO(3) growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K') was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO(2)-HCO(3) as a consequence of regulating electron sink metabolism.

Journal Article↗

PubMed1991

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.

Animals↗