23 results on '"Cuskin, Fiona"'
Search Results
2. Mechanisms by which glycoside hydrolases recognize plant, bacterial and yeast polysaccharides
- Author
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Cuskin, Fiona Marie
- Subjects
572 - Abstract
The deconstruction of complex carbohydrates by glycoside hydrolases requires extensive enzyme consortia in which specificity is often conferred by accessory modules and domains that are distinct from the active site. The diverse mechanisms of substrate recognition were explored in this thesis using selected yeast, bacterial and plant polysaccharides as example substrates. Carbohydrate binding modules (CBM) are non-catalytic modules that enhance the catalytic activity of their glycoside hydrolase counterparts through binding to polysaccharide. Normally CBMs are found attached to glycoside hydrolases that target insoluble recalcitrant substrates resulting in a moderate, 2-5 fold, potentiation in enzyme activity. A CBM, defined herein as CBMX40, is found at the C-terminal of a glycoside hydrolase family (GH) 32 enzyme, SacC, which displays exo-levanase activity. CBMX40 binds the non-reducing end of the levan chain targeting the disaccharide fructose--fructose unit. Removal of CBMX40 results in a >100-fold decrease in catalytic activity against levan, compared to the full length native enzyme. The truncated SacC catalytic domain acts as a non-specific exo-β-fructosidase displaying similar activity on β2,1- (inulin) and β2,6-linked fructose polymers, both polysaccharides and oligosaccharides. When CBMX40 was fused to a non-related exo-β-fructosidase, BT 3082, it conferred exo-levanase specificity on the enzyme. Thus CBMX40 is not only able to enhance catalytic activity but is also able to confer catalytic specificity. This led to the hypothesis that the CBM and the active site of the enzyme bind to different terminal residues of branched fructans such as levan. This results in enhanced affinity through avidity effects leading to the potentiation of catalytic activity. The gut bacterium Bacteroides thetaiotaomicron contributes to the maintenance of a healthy human gut. B. thetaiotaomicron is able to acquire and utilise complex carbohydrates that are not attacked by the intestinal enzymes of the host. B. thetaiotaomicron dedicates a large proportion of its genome to glycan degradation with a large expansion of α-mannan degrading enzymes. The B. thetaiotaomicron genome encodes 23 GH92 α-mannanosidases and 10 GH76 α-mannanases. While GH92 has recently been characterised the activities displayed by GH76 relies on the characterization of a single enzyme in this family. B. thetaiotaomicron organises the genes required to sense, degrade, transport and utilise specific complex glycans into genetic clusters defined as Polysaccharide Utilisation Loci (PULs). Transcriptomics revealed that two PULs are up regulated in response to yeast mannan, PUL 36 and PUL 68. These PULs contain both GH76 enzymes along with GH92 enzymes and other CAZy annotated enzymes. Biochemical analysis of the GH76 enzymes found in the two PULs show they are α1, 6 mannanases capable of hydrolysing the α1, 6 mannan backbone of yeast mannan, with the putative periplasmic enzymes generating small oligosaccharides, while the surface mannanases releasing larger products. The three GH92 enzymes encoded by the two PULs have been shown to remove α1, 2 and α1, 3 linked mannose branches from yeast mannan polysaccharide. In addition PUL 68 also encodes a phosphatase that removes the phosphate from mannose-6-phosphate and glucose-6-phosphate but not from intact mannan. Therefore, this study describes the ability of B. thetaiotaomicron to target and degrade yeast α-mannans. The GH5 enzyme CtXyl5A from Clostridium thermocellum is an arabinoxylan specific xylanase that contains a GH5 catalytic module appended to several CBMs. The apo structure of the GH5 catalytic module appended to a family 6 CBM reveals a large pocket abutted to the -1 subsite of the active site. This pocket was thought to bind the arabinose decoration appended to the O3 of the xylan backbone. Here mutational and structural studies showed that the fulfilment of arabinose is this pocket is the key specificity determinant for the novel arabinoxylanase activity. Significantly the bound arabinose displayed a pyranose conformation, rather than a furanose structure which is the typical conformation adopted by arabinose side chains in arabinoxylans. This structural information suggests that CtXyl5A may be able to exploit side chains other than arabinofuranose residues as substrate specificity determinants.
- Published
- 2013
3. Glycan degradation writ large in the ocean
- Author
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Cuskin, Fiona and Lowe, Elisabeth C.
- Published
- 2020
- Full Text
- View/download PDF
4. How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity
- Author
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Cuskin, Fiona, Flint, James E., Gloster, Tracey M., Morland, Carl, Baslé, Arnaud, Henrissat, Bernard, Coutinho, Pedro M., Strazzulli, Andrea, Solovyova, Alexandra S., Davies, Gideon J., and Gilbert, Harry J.
- Published
- 2012
5. Analysis of Glycans in a Burnt-On/Baked-On (Bobo) Model Food Soil Using Microarray Polymer Profiling (Mapp) and Immunofluorescence Microscopy
- Author
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Bakshani, Cassie Rose, primary, Cuskin, Fiona, additional, Lant, Neil, additional, Yau, Hamish, additional, Willats, William, additional, and Burgess, Grant, additional
- Published
- 2022
- Full Text
- View/download PDF
6. Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism
- Author
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Cuskin, Fiona, Lowe, Elisabeth C., Temple, Max J., Zhu, Yanping, Cameron, Elizabeth A., Pudlo, Nicholas A., Porter, Nathan T., Urs, Karthik, Thompson, Andrew J., Cartmell, Alan, Rogowski, Artur, Hamilton, Brian S., Chen, Rui, Tolbert, Thomas J., Piens, Kathleen, Bracke, Debby, Vervecken, Wouter, Hakki, Zalihe, Speciale, Gaetano, Munōz-Munōz, Jose L., Day, Andrew, Peña, Maria J., McLean, Richard, Suits, Michael D., Boraston, Alisdair B., Atherly, Todd, Ziemer, Cherie J., Williams, Spencer J., Davies, Gideon J., Abbott, Wade D., Martens, Eric C., and Gilbert, Harry J.
- Published
- 2015
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7. Structure and function of a glycoside hydrolase family 8 endoxylanase from Teredinibacter turnerae
- Author
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Fowler, Claire A, Hemsworth, Glyn R, Cuskin, Fiona, Hart, Sam, Turkenburg, Johan, Gilbert, Harry J., Walton, Paul H, and Davies, Gideon J
- Subjects
Gram-Negative Facultatively Anaerobic Rods ,animal structures ,shipworms ,Endo-1,4-beta Xylanases ,biomass ,Glycoside Hydrolases ,Protein Conformation ,food and beverages ,Plants ,Research Papers ,biofuels ,Kinetics ,Bacterial Proteins ,Polysaccharides ,marine polysaccharides ,Teredinibacter turnerae ,Plant Cells ,glycoside hydrolase ,Xylans ,cellulolytic enzymes ,Gammaproteobacteria - Abstract
The symbionts of marine shipworms provide a rich reservoir of potential carbohydrate-active enzymes. Here, the 1.5 Å resolution three-dimensional structure of a T. turnerae GH8 xylanase is revealed and its potential in biomass degradation is highlighted., The biological conversion of lignocellulosic matter into high-value chemicals or biofuels is of increasing industrial importance as the sector slowly transitions away from nonrenewable sources. Many industrial processes involve the use of cellulolytic enzyme cocktails – a selection of glycoside hydrolases and, increasingly, polysaccharide oxygenases – to break down recalcitrant plant polysaccharides. ORFs from the genome of Teredinibacter turnerae, a symbiont hosted within the gills of marine shipworms, were identified in order to search for enzymes with desirable traits. Here, a putative T. turnerae glycoside hydrolase from family 8, hereafter referred to as TtGH8, is analysed. The enzyme is shown to be active against β-1,4-xylan and mixed-linkage (β-1,3,β-1,4) marine xylan. Kinetic parameters, obtained using high-performance anion-exchange chromatography with pulsed amperometric detection and 3,5-dinitrosalicyclic acid reducing-sugar assays, show that TtGH8 catalyses the hydrolysis of β-1,4-xylohexaose with a k cat/K m of 7.5 × 107 M −1 min−1 but displays maximal activity against mixed-linkage polymeric xylans, hinting at a primary role in the degradation of marine polysaccharides. The three-dimensional structure of TtGH8 was solved in uncomplexed and xylobiose-, xylotriose- and xylohexaose-bound forms at approximately 1.5 Å resolution; the latter was consistent with the greater k cat/K m for hexasaccharide substrates. A 2,5 B boat conformation observed in the −1 position of bound xylotriose is consistent with the proposed conformational itinerary for this class of enzyme. This work shows TtGH8 to be effective at the degradation of xylan-based substrates, notably marine xylan, further exemplifying the potential of T. turnerae for effective and diverse biomass degradation.
- Published
- 2018
8. The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans
- Author
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Labourel, Aurore, Crouch, Lucy I., Bras, Joana L. A., Jackson, Adam, Rogowski, Artur, Gray, Joseph, Yadav, Madhav P., Henrissat, Bernard, Fontes, Carlos M. G. A., Gilbert, Harry J., Najmudin, Shabir, Basle, Arnaud, Cuskin, Fiona, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), European Research Council [322820], Biotechnology and Biological Research Council [BB/K020358/1, BB/K001949/1], Wellcome Trust [RES/0120/7613], Agence Nationale de la Recherche [ANR 12-BIME-0006-01], and Fundacao para a Ciencia e Tecnologia [PTDC/BIAPRO/103980/2008, PTDC/BIAMIC/5947/2014]
- Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,cellulosome ,crystallography ,enzyme kinetics ,enzyme mechanism ,glycoside hydrolase ,Crystallography, X-Ray ,[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,Clostridium thermocellum ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Xylosidases ,Bacterial Proteins ,Protein Domains ,Enzymology ,Xylans ,ComputingMilieux_MISCELLANEOUS - Abstract
The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of -1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp--1,4-Xylp--1,4-Xylp-[-1,3-Araf]--1,4-Xylp showed that the Araf decoration linked O-3 to the xylose in the active site is located in the pocket (-2* subsite) that abuts onto the catalytic center. The -2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu(68), Tyr(92), or Asn(139), which interact with arabinose and xylose side chains at the -2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.
- Published
- 2016
9. A Large Polysaccharide Produced by Helicobacter hepaticus Induces an Anti-inflammatory Gene Signature in Macrophages
- Author
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Danne, Camille, primary, Ryzhakov, Grigory, additional, Martínez-López, Maria, additional, Ilott, Nicholas Edward, additional, Franchini, Fanny, additional, Cuskin, Fiona, additional, Lowe, Elisabeth C., additional, Bullers, Samuel J., additional, Arthur, J. Simon C., additional, and Powrie, Fiona, additional
- Published
- 2017
- Full Text
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10. A Bacteroidetes locus dedicated to fungal 1,6-β-glucan degradation: Unique substrate conformation drives specificity of the key endo-1,6-β-glucanase
- Author
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Temple, Max J., primary, Cuskin, Fiona, additional, Baslé, Arnaud, additional, Hickey, Niall, additional, Speciale, Gaetano, additional, Williams, Spencer J., additional, Gilbert, Harry J., additional, and Lowe, Elisabeth C., additional
- Published
- 2017
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11. Diverse specificity of cellulosome attachment to the bacterial cell surface
- Author
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Brás, Joana L. A., primary, Pinheiro, Benedita A., additional, Cameron, Kate, additional, Cuskin, Fiona, additional, Viegas, Aldino, additional, Najmudin, Shabir, additional, Bule, Pedro, additional, Pires, Virginia M. R., additional, Romão, Maria João, additional, Bayer, Edward A., additional, Spencer, Holly L., additional, Smith, Steven, additional, Gilbert, Harry J., additional, Alves, Victor D., additional, Carvalho, Ana Luísa, additional, and Fontes, Carlos M. G. A., additional
- Published
- 2016
- Full Text
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12. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism
- Author
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Jin, Yi, primary, Petricevic, Marija, additional, John, Alan, additional, Raich, Lluís, additional, Jenkins, Huw, additional, Portela De Souza, Leticia, additional, Cuskin, Fiona, additional, Gilbert, Harry J., additional, Rovira, Carme, additional, Goddard-Borger, Ethan D., additional, Williams, Spencer J., additional, and Davies, Gideon J., additional
- Published
- 2016
- Full Text
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13. Structure and function of a glycoside hydrolase family 8 endoxylanase from Teredinibacter turnerae.
- Author
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Fowler, Claire A., Hemsworth, Glyn R., Cuskin, Fiona, Hart, Sam, Turkenburg, Johan, Gilbert, Harry J., Walton, Paul H., and Davies, Gideon J.
- Subjects
GLYCOSIDASES ,XYLANASES ,BIOMASS energy - Abstract
The biological conversion of lignocellulosic matter into high‐value chemicals or biofuels is of increasing industrial importance as the sector slowly transitions away from nonrenewable sources. Many industrial processes involve the use of cellulolytic enzyme cocktails – a selection of glycoside hydrolases and, increasingly, polysaccharide oxygenases – to break down recalcitrant plant polysaccharides. ORFs from the genome of Teredinibacter turnerae, a symbiont hosted within the gills of marine shipworms, were identified in order to search for enzymes with desirable traits. Here, a putative T. turnerae glycoside hydrolase from family 8, hereafter referred to as TtGH8, is analysed. The enzyme is shown to be active against β‐1,4‐xylan and mixed‐linkage (β‐1,3,β‐1,4) marine xylan. Kinetic parameters, obtained using high‐performance anion‐exchange chromatography with pulsed amperometric detection and 3,5‐dinitrosalicyclic acid reducing‐sugar assays, show that TtGH8 catalyses the hydrolysis of β‐1,4‐xylohexaose with a k
cat /Km of 7.5 × 107 M−1 min−1 but displays maximal activity against mixed‐linkage polymeric xylans, hinting at a primary role in the degradation of marine polysaccharides. The three‐dimensional structure of TtGH8 was solved in uncomplexed and xylobiose‐, xylotriose‐ and xylohexaose‐bound forms at approximately 1.5 Å resolution; the latter was consistent with the greater kcat /Km for hexasaccharide substrates. A2,5 B boat conformation observed in the −1 position of bound xylotriose is consistent with the proposed conformational itinerary for this class of enzyme. This work shows TtGH8 to be effective at the degradation of xylan‐based substrates, notably marine xylan, further exemplifying the potential of T. turnerae for effective and diverse biomass degradation. [ABSTRACT FROM AUTHOR]- Published
- 2018
- Full Text
- View/download PDF
14. The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan
- Author
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Cuskin, Fiona, primary, Baslé, Arnaud, additional, Ladevèze, Simon, additional, Day, Alison M., additional, Gilbert, Harry J., additional, Davies, Gideon J., additional, Potocki-Véronèse, Gabrielle, additional, and Lowe, Elisabeth C., additional
- Published
- 2015
- Full Text
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15. Coevolution of yeast mannan digestion: Convergence of the civilized human diet, distal gut microbiome, and host immunity
- Author
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Abbott, D Wade, primary, Martens, Eric C, additional, Gilbert, Harry J, additional, Cuskin, Fiona, additional, and Lowe, Elisabeth C, additional
- Published
- 2015
- Full Text
- View/download PDF
16. Erratum: Corrigendum: Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism
- Author
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Cuskin, Fiona, primary, Lowe, Elisabeth C., additional, Temple, Max J., additional, Zhu, Yanping, additional, Cameron, Elizabeth A., additional, Pudlo, Nicholas A., additional, Porter, Nathan T., additional, Urs, Karthik, additional, Thompson, Andrew J., additional, Cartmell, Alan, additional, Rogowski, Artur, additional, Hamilton, Brian S., additional, Chen, Rui, additional, Tolbert, Thomas J., additional, Piens, Kathleen, additional, Bracke, Debby, additional, Vervecken, Wouter, additional, Hakki, Zalihe, additional, Speciale, Gaetano, additional, Munōz-Munōz, Jose L., additional, Day, Andrew, additional, Peña, Maria J., additional, McLean, Richard, additional, Suits, Michael D., additional, Boraston, Alisdair B., additional, Atherly, Todd, additional, Ziemer, Cherie J., additional, Williams, Spencer J., additional, Davies, Gideon J., additional, Abbott, D. Wade, additional, Martens, Eric C., additional, and Gilbert, Harry J., additional
- Published
- 2015
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17. Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiontBacteroides thetaiotaomicron
- Author
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Thompson, Andrew J., primary, Cuskin, Fiona, additional, Spears, Richard J., additional, Dabin, Jerome, additional, Turkenburg, Johan P., additional, Gilbert, Harry J., additional, and Davies, Gideon J., additional
- Published
- 2015
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18. Crystallization and preliminary X-ray analysis of the bacillaene synthasetrans-acting acyltransferase PksC
- Author
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Cuskin, Fiona, primary, Solovyova, Alexandra S., additional, Lewis, Richard J., additional, and Race, Paul R., additional
- Published
- 2011
- Full Text
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19. Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiont Bacteroides thetaiotaomicron.
- Author
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Thompson, Andrew J., Cuskin, Fiona, Spears, Richard J., Dabin, Jerome, Turkenburg, Johan P., Gilbert, Harry J., and Davies, Gideon J.
- Subjects
- *
HOMOLOGY (Biology) , *BACTEROIDES thetaiotaomicron , *ANTIGENS , *IMMUNE system , *LOCUS (Genetics) , *POLYSACCHARIDES - Abstract
The large bowel microbiota, a complex ecosystem resident within the gastrointestinal tract of all human beings and large mammals, functions as an essential, nonsomatic metabolic organ, hydrolysing complex dietary polysaccharides and modulating the host immune system to adequately tolerate ingested antigens. A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host. The immense ability of B. thetaiotaomicron as a `glycan specialist' resides in its enormous array of carbohydrate-active enzymes, many of which are arranged into polysaccharide-utilization loci (PULs) that are able to degrade sugar polymers that are often inaccessible to other gut residents, notably α-mannan. The B. thetaiotaomicron genome encodes ten putative α-mannanases spread across various PULs; however, little is known about the activity of these enzymes or the wider implications of α-mannan metabolism for the health of both the microbiota and the host. In this study, SAD phasing of a selenomethionine derivative has been used to investigate the structure of one such B. thetaiotaomicron enzyme, BT2949, which belongs to the GH76 family of α-mannanases. BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members. Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
20. Crystallization and preliminary X-ray analysis of the bacillaene synthase trans-acting acyltransferase PksC.
- Author
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Cuskin, Fiona, Solovyova, Alexandra S., Lewis, Richard J., and Race, Paul R.
- Subjects
- *
CRYSTALLIZATION , *ACYLTRANSFERASES , *BACILLUS subtilis , *NONRIBOSOMAL peptide synthetases , *BIOSYNTHESIS - Abstract
The antibiotic bacillaene is biosynthesized in Bacillus subtilis by a hybrid type 1 modular polyketide synthase/nonribosomal peptide synthetase of the trans-acyltransferase ( trans-AT) class. Within this system, the essential acyl-group loading activity is provided by the action of three free-standing trans-acting acyltransferases. Here, the recombinant expression, purification and crystallization of the bacillaene synthase trans-acting acyltransferase PksC are reported. A diffraction data set has been collected from a single PksC crystal to 1.44 Å resolution and the crystal was found to belong to the orthorhombic space group P212121. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
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21. Corrigendum: Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.
- Author
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Cuskin, Fiona, Lowe, Elisabeth C., Temple, Max J., Zhu, Yanping, Cameron, Elizabeth A., Pudlo, Nicholas A., Porter, Nathan T., Urs, Karthik, Thompson, Andrew J., Cartmell, Alan, Rogowski, Artur, Hamilton, Brian S., Chen, Rui, Tolbert, Thomas J., Piens, Kathleen, Bracke, Debby, Vervecken, Wouter, Hakki, Zalihe, Speciale, Gaetano, and Munōz-Munōz, Jose L.
- Subjects
- *
BACTEROIDES , *YEAST - Abstract
A correction to the article "Human Gut Bacteroidetes Can Utilize Yeast Mannan Through A Selfish Mechanism" by Fiona Cuskin, Max J. Temple and Yanping Zhu published in "Nature" Vol 517 2015 issue is presented.
- Published
- 2015
- Full Text
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22. How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity
- Author
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Fiona Cuskin, a, b, 1 James E. Flint, 1 Tracey M. Gloster, c, 1, 2 Carl Morland, a Arnaud Baslé, a Bernard Henrissat, d Pedro M. Coutinho, d Andrea Strazzulli, e Alexandra S. Solovyova, a Gideon J. Davies, Harry J. Gilberta, 3, Cuskin, Fiona, Flint, James E, Gloster, Tracey M, Morland, Carl, Baslé, Arnaud, Henrissat, Bernard, Coutinho, Pedro M, Strazzulli, Andrea, Solovyova, Alexandra S, Davies, Gideon J, and Gilbert, Harry J.
- Subjects
Glycoside Hydrolase ,Oligosaccharides ,Plasma protein binding ,Bacillus subtilis ,Crystallography, X-Ray ,Ligands ,Catalysi ,Oligosaccharide ,Lectins ,Bacteroides ,Glycoside hydrolase ,Polysaccharide ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Chemistry ,Biological Sciences ,isothermal titration calorimetry ,Enzymes ,Biochemistry ,Hydrophobic and Hydrophilic Interactions ,Protein Binding ,Carbohydrate ,Glycoside Hydrolases ,Stereochemistry ,Carbohydrates ,Ligand ,Calorimetry ,Catalysis ,Fructan ,Hydrophobic and Hydrophilic Interaction ,Biofuel ,Polysaccharides ,Hydrolase ,Bacillus subtili ,X-ray crystallography ,Kinetic ,Substrate (chemistry) ,biology.organism_classification ,Fructans ,Protein Structure, Tertiary ,Kinetics ,Enzyme ,Bacteroide ,Models, Chemical ,Biofuels ,prebiotics ,Lectin ,Function (biology) - Abstract
Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two- to fivefold elevation in the activity of endo-acting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 ( Bs CBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of Bs CBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of Bs CBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1– and β-2,6–linked fructans and their respective fructooligosaccharides. Conversely, appending Bs CBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron , confers exolevanase activity on the enzyme. We propose that Bs CBM66 confers specificity for levan, a branched fructan, through an “avidity” mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme.
- Published
- 2012
23. The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans.
- Author
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Labourel A, Crouch LI, Brás JL, Jackson A, Rogowski A, Gray J, Yadav MP, Henrissat B, Fontes CM, Gilbert HJ, Najmudin S, Baslé A, and Cuskin F
- Subjects
- Crystallography, X-Ray, Protein Domains, Bacterial Proteins chemistry, Clostridium thermocellum enzymology, Xylans chemistry, Xylosidases chemistry
- Abstract
The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O
3 to the xylose in the active site is located in the pocket (-2* subsite) that abuts onto the catalytic center. The -2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68 , Tyr92 , or Asn139 , which interact with arabinose and xylose side chains at the -2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2016
- Full Text
- View/download PDF
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