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  • 1
    Online Resource
    Online Resource
    Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis
    Proceedings of the National Academy of Sciences ; 2011
    In:  Proceedings of the National Academy of Sciences Vol. 108, No. 13 ( 2011-03-29), p. 5237-5242
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 13 ( 2011-03-29), p. 5237-5242
    Abstract: Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall–degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated Ct Cel124. The protein was shown to be an endo -acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo -cellulase. The crystal structure of Ct Cel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1015006108
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2011
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 2
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    Online Resource
    Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors
    Proceedings of the National Academy of Sciences ; 2010
    In:  Proceedings of the National Academy of Sciences Vol. 107, No. 43 ( 2010-10-26), p. 18646-18651
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 107, No. 43 ( 2010-10-26), p. 18646-18651
    Abstract: Clostridium thermocellum produces a highly efficient cellulolytic extracellular complex, termed the cellulosome, for hydrolyzing plant cell wall biomass. The composition of the cellulosome is affected by the presence of extracellular polysaccharides; however, the regulatory mechanism is unknown. Recently, we have identified in C. thermocellum a set of putative σ and anti-σ factors that include extracellular polysaccharide-sensing components [Kahel-Raifer et al. (2010) FEMS Microbiol Lett 308:84–93]. These factor-encoding genes are homologous to the Bacillus subtilis bicistronic operon sigI-rsgI , which encodes for an alternative σ I factor and its cognate anti-σ I regulator RsgI that is functionally regulated by an extracytoplasmic signal. In this study, the binding of C. thermocellum putative anti-σ I factors to their corresponding σ factors was measured, demonstrating binding specificity and dissociation constants in the range of 0.02 to 1 μM. Quantitative real-time RT-PCR measurements revealed three- to 30-fold up-expression of the alternative σ factor genes in the presence of cellulose and xylan, thus connecting their expression to direct detection of their extracellular polysaccharide substrates. Cellulosomal genes that are putatively regulated by two of these σ factors, σ I1 or σ I6 , were identified based on the sequence similarity of their promoters. The ability of σ I1 to direct transcription from the sigI1 promoter and from the promoter of celS (encodes the family 48 cellulase) was demonstrated in vitro by runoff transcription assays. Taken together, the results reveal a regulatory mechanism in which alternative σ factors are involved in regulating the cellulosomal genes via an external carbohydrate-sensing mechanism.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1012175107
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2010
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 3
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    Online Resource
    Single-molecule dissection of the high-affinity cohesin–dockerin complex
    Proceedings of the National Academy of Sciences ; 2012
    In:  Proceedings of the National Academy of Sciences Vol. 109, No. 50 ( 2012-12-11), p. 20431-20436
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 50 ( 2012-12-11), p. 20431-20436
    Abstract: Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin–dockerin rupture forces ( 〉 120 pN) are among the highest reported for a receptor–ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop–helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin’s critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein–protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1211929109
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2012
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 4
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    Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation
    Proceedings of the National Academy of Sciences ; 2014
    In:  Proceedings of the National Academy of Sciences Vol. 111, No. 25 ( 2014-06-24), p. 9109-9114
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 111, No. 25 ( 2014-06-24), p. 9109-9114
    Abstract: Efficient conversion of cellulose into soluble sugars is a key technological bottleneck limiting efficient production of plant-derived biofuels and chemicals. In nature, the process is achieved by the action of a wide range of cellulases and associated enzymes. In aerobic microrganisms, cellulases are secreted as free enzymes. Alternatively, in certain anaerobic microbes, cellulases are assembled into large multienzymes complexes, termed “cellulosomes,” which allow for efficient hydrolysis of cellulose. Recently, it has been shown that enzymes classified as lytic polysaccharide monooxygenases (LPMOs) were able to strongly enhance the activity of cellulases. However, LPMOs are exclusively found in aerobic organisms and, thus, cannot benefit from the advantages offered by the cellulosomal system. In this study, we designed several dockerin-fused LPMOs based on enzymes from the bacterium Thermobifida fusca . The resulting chimeras exhibited activity levels on microcrystalline cellulose similar to that of the wild-type enzymes. The dockerin moieties of the chimeras were demonstrated to be functional and to specifically bind to their corresponding cohesin partner. The chimeric LPMOs were able to self-assemble in designer cellulosomes alongside an endo- and an exo-cellulase also converted to the cellulosomal mode. The resulting complexes showed a 1.7-fold increase in the release of soluble sugars from cellulose, compared with the free enzymes, and a 2.6-fold enhancement compared with free cellulases without LPMO enhancement. These results highlight the feasibility of the conversion of LPMOs to the cellulosomal mode, and that these enzymes can benefit from the proximity effects generated by the cellulosome architecture.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1404148111
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2014
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 5
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    Structural basis of Clostridium perfringens toxin complex formation
    Proceedings of the National Academy of Sciences ; 2008
    In:  Proceedings of the National Academy of Sciences Vol. 105, No. 34 ( 2008-08-26), p. 12194-12199
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 105, No. 34 ( 2008-08-26), p. 12194-12199
    Abstract: The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the μ-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction ( K a = 1.44 × 10 11 M −1 ) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The μ-toxin dockerin module in this complex is positioned ≈180° relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0803154105
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2008
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 6
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    Evidence for a dual binding mode of dockerin modules to cohesins
    Proceedings of the National Academy of Sciences ; 2007
    In:  Proceedings of the National Academy of Sciences Vol. 104, No. 9 ( 2007-02-27), p. 3089-3094
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 104, No. 9 ( 2007-02-27), p. 3089-3094
    Abstract: The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived “type I dockerin” modules with the multiple “cohesin” modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180° relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0611173104
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2007
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 7
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    Plant Cell Wall Breakdown by Anaerobic Microorganisms from the Mammalian Digestive Tract
    Wiley ; 2008
    In:  Annals of the New York Academy of Sciences Vol. 1125, No. 1 ( 2008-03), p. 280-288
    Details
    In: Annals of the New York Academy of Sciences, Wiley, Vol. 1125, No. 1 ( 2008-03), p. 280-288
    Abstract: Degradation of lignocellulosic plant material in the mammalian digestive tract is accomplished by communities of anaerobic microorganisms that exist in symbiotic association with the host. Catalytic domains and substrate‐binding modules concerned with plant polysaccharide degradation are found in a variety of anaerobic bacteria, fungi, and protozoa from the mammalian gut. The organization of plant cell wall–degrading enzymes, however, varies widely. The cellulolytic gram‐positive bacterium Ruminococcus flavefaciens produces an elaborate cellulosomal enzyme complex that is anchored to the bacterial cell wall; assembly of the complex involves at least five different dockerin:cohesin specificities, and the R. flavefaciens genome encodes at least 180 dockerin‐containing proteins that encompass a wide array of catalytic and binding activities. On the other hand, in the cellulolytic protozoan, Polyplastron multivesiculatum , individual plant cell wall–degrading enzymes appear to be secreted into food vacuoles, while the gram‐negative bacterium Prevotella bryantii appears to possess a sequestration‐type system for the utilization of soluble xylans. The system that is employed for polysaccharide utilization must play a major role in defining the ecological niche that each organism occupies within a complex gut community. 16S rRNA analyses are also revealing uncultured bacterial species closely adherent to fibrous substrates in the rumen and in the large intestine of animals and humans. The true complexity, both at a single organism and community level, of the microbial enzyme systems that allow animals to digest plant material is beginning to become apparent.
    Type of Medium: Online Resource
    ISSN: 0077-8923 , 1749-6632
    URL: Issue
    URL: Article
    DOI: 10.1196/nyas.2008.1125.issue-1
    DOI: 10.1196/annals.1419.022
    RVK:
    TD 7650
    Language: English
    Publisher: Wiley
    Publication Date: 2008
    detail.hit.zdb_id: 2834079-6
    detail.hit.zdb_id: 211003-9
    detail.hit.zdb_id: 2071584-5
    SSG: 11
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  • 8
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    Online Resource
    Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome
    Proceedings of the National Academy of Sciences ; 2012
    In:  Proceedings of the National Academy of Sciences Vol. 109, No. 26 ( 2012-06-26), p. 10298-10303
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 26 ( 2012-06-26), p. 10298-10303
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1202747109
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2012
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 9
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    Online Resource
    Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum
    Proceedings of the National Academy of Sciences ; 2018
    In:  Proceedings of the National Academy of Sciences Vol. 115, No. 48 ( 2018-11-27)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 115, No. 48 ( 2018-11-27)
    Abstract: Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum . We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin–cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as “beads on a string,” we propose that cohesin–cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1809283115
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2018
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 10
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    Online Resource
    Structural elements responsible for conversion of streptavidin to a pseudoenzyme
    Proceedings of the National Academy of Sciences ; 2004
    In:  Proceedings of the National Academy of Sciences Vol. 101, No. 16 ( 2004-04-20), p. 5916-5921
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 101, No. 16 ( 2004-04-20), p. 5916-5921
    Abstract: Avidin enhances the hydrolysis of biotinyl p -nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p -nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values 〉 8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0308541101
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2004
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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