40 results on '"Hemsworth, Glyn R."'
Search Results
2. Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae
- Author
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Fowler, Claire. A., Sabbadin, Federico, Ciano, Luisa, Hemsworth, Glyn R., Elias, Luisa, Bruce, Neil, McQueen-Mason, Simon, Davies, Gideon J., and Walton, Paul H.
- Published
- 2019
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- View/download PDF
3. Spectroscopic and computational insight into the activation of O 2 by the mononuclear Cu center in polysaccharide monooxygenases
- Author
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Kjaergaard, Christian H., Qayyum, Munzarin F., Wong, Shaun D., Xu, Feng, Hemsworth, Glyn R., Walton, Daniel J., Young, Nigel A., Davies, Gideon J., Walton, Paul H., Johansen, Katja Salomon, Hodgson, Keith O., Hedman, Britt, and Solomon, Edward I.
- Published
- 2014
4. Revisiting the role of electron donors in lytic polysaccharide monooxygenase biochemistry.
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Hemsworth, Glyn R.
- Published
- 2023
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5. A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes
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Larsbrink, Johan, Rogers, Theresa E., Hemsworth, Glyn R., McKee, Lauren S., Tauzin, Alexandra S., Spadiut, Oliver, Klinter, Stefan, Pudlo, Nicholas A., Urs, Karthik, Koropatkin, Nicole M., Creagh, A. Louise, Haynes, Charles A., Kelly, Amelia G., Cederholm, Stefan Nilsson, Davies, Gideon J., Martens, Eric C., and Brumer, Harry
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Metabolism -- Genetic aspects ,Microbiota (Symbiotic organisms) -- Genetic aspects -- Physiological aspects ,Xyloglucans -- Genetic aspects -- Physiological aspects ,Quantitative trait loci -- Physiological aspects ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables (1). Owing to the paucity of alimentary enzymes encoded by the human genome (2), our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut (3,4). The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides (5,6) whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear (1,7,8). Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre maybe mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health (9-12)., Despite our omnivory, a census of the glycoside hydrolases (GHs) encoded by the human genome indicates that our inherent ability to digest carbohydrates is restricted to starch and simple saccharides [...]
- Published
- 2014
6. The structure of Escherichia coli ExoIX—implications for DNA binding and catalysis in flap endonucleases
- Author
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Anstey-Gilbert, Christopher S., Hemsworth, Glyn R., Flemming, Claudia S., Hodskinson, Michael R. G., Zhang, Jing, Sedelnikova, Svetlana E., Stillman, Timothy J., Sayers, Jon R., and Artymiuk, Peter J.
- Published
- 2013
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7. Crystal structure of the small GTPase Arl6/BBS3 from Trypanosoma brucei
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Hemsworth, Glyn R., Price, Helen P., Smith, Deborah F., and Wilson, Keith S.
- Published
- 2013
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8. An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion
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Sabbadin, Federico, Hemsworth, Glyn R., Ciano, Luisa, Henrissat, Bernard, Dupree, Paul, Tryfona, Theodora, Marques, Rita D. S., Sweeney, Sean T., Besser, Katrin, Elias, Luisa, Pesante, Giovanna, Li, Yi, Dowle, Adam A., Bates, Rachel, Gomez, Leonardo D., Simister, Rachael, Davies, Gideon J., Walton, Paul H., Bruce, Neil C., McQueen-Mason, Simon J., University of York [York, UK], University of Leeds, 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), University of Cambridge [UK] (CAM), Hemsworth, Glyn R [0000-0002-8226-1380], Ciano, Luisa [0000-0002-1667-0856], Dupree, Paul [0000-0001-9270-6286], Sweeney, Sean T [0000-0003-2673-9578], Davies, Gideon J [0000-0002-7343-776X], Walton, Paul H [0000-0002-1152-1480], Apollo - University of Cambridge Repository, Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), UK Biotechnology and Biological Sciences Research Council [BB/L001926/1], Yorkshire Forward, Northern Way Initiative, and EPSRC [EP/K039660/1, EP/M028127/1]
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Models, Molecular ,Proteomics ,Insecta ,Science ,Chitin ,Genes, Insect ,Article ,Mixed Function Oxygenases ,Evolution, Molecular ,Polysaccharides ,Animals ,Biomass ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,lcsh:Science ,Cellulose ,Arthropods ,Phylogeny ,food and beverages ,Enzymes ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Biodegradation, Environmental ,Insect Proteins ,lcsh:Q - Abstract
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases., LPMOs catalyze the oxidative breakdown of polysaccharides, thereby facilitating biomass degradation. By analyzing the digestive proteome of firebrats, the authors here identify a yet uncharacterized LPMO family and provide phylogenetic, structural and biochemical insights into its origin and functions.
- Published
- 2018
9. A cell-surface GH9 endo-glucanase coordinates with surface glycan binding proteins to mediate xyloglucan uptake in the gut symbiont Bacteroides ovatus
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Foley, Matthew H, Dejean, Guillaume, Hemsworth, Glyn R, Martens, Eric C, Davies, Gideon J, Brumer, Harry, and Koropatkin, Nicole M
- Published
- 2019
10. Structure and function of a glycoside hydrolase family 8 endoxylanase from Teredinibacter turnerae
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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
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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
11. C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.
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Branch, Jessie, Rajagopal, Badri S., Paradisi, Alessandro, Yates, Nick, Lindley, Peter J., Smith, Jake, Hollingsworth, Kristian, Turnbull, W. Bruce, Henrissat, Bernard, Parkin, Alison, Berry, Alan, and Hemsworth, Glyn R.
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MONOOXYGENASES ,ELECTRON sources ,LYSINS ,ELECTRON donors ,LIGNOCELLULOSE ,ENERGY futures ,CELLULASE ,CELLULOSE synthase - Abstract
The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H
2 O2 production from O2 , and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria. [ABSTRACT FROM AUTHOR]- Published
- 2021
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12. Insights from semi-oriented EPR spectroscopy studies into the interaction of lytic polysaccharide monooxygenases with cellulose.
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Ciano, Luisa, Paradisi, Alessandro, Hemsworth, Glyn R., Tovborg, Morten, Davies, Gideon J., and Walton, Paul H.
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ELECTRON paramagnetic resonance spectroscopy ,MONOOXYGENASES ,CELLULOSE ,MAGNETIC spectrometer ,CELERY ,CELLULOSE synthase - Abstract
Probing the detailed interaction between lytic polysaccharide monooxygenases (LPMOs) and their polysaccharide substrates is key to revealing further insights into the mechanism of action of this class of enzymes on recalcitrant biomass. This investigation is somewhat hindered, however, by the insoluble nature of the substrates, which precludes the use of most optical spectroscopic techniques. Herein, we report a new semi-oriented EPR method which evaluates directly the binding of cellulose-active LPMOs to crystalline cellulose. We make use of the intrinsic order of cellulose fibres in Apium graveolens (celery) to orient the LPMO with respect to the magnetic field of an EPR spectrometer. The subsequent angle-dependent changes observed in the EPR spectra can then be related to the orientation of the g matrix principal directions with respect to the magnetic field of the spectrometer and, hence, to the binding of the enzyme onto the cellulose fibres. This method, which does not require specific modification of standard CW-EPR equipment, can be used as a general procedure to investigate LPMO–cellulose interactions. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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13. Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut
- Author
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Hemsworth, Glyn R., Thompson, Andrew J., Stepper, Judith, Sobala, Łukasz F., Coyle, Travis, Larsbrink, Johan, Spadiut, Oliver, Goddard-Borger, Ethan D., Stubbs, Keith A., Brumer, Harry, and Davies, Gideon J.
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xyloglucan ,lcsh:Biology (General) ,glycoside hydrolases ,polysaccharide utilization loci ,lcsh:QH301-705.5 - Abstract
The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta) genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-L-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.
- Published
- 2016
14. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase
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Lo Leggio, Leila, Simmons, Thomas J., Poulsen, Jens Christian N, Frandsen, Kristian E H, Hemsworth, Glyn R., Stringer, Mary A., Von Freiesleben, Pernille, Tovborg, Morten, Johansen, Katja S., De Maria, Leonardo, Harris, Paul V., Soong, Chee Leong, Dupree, Paul, Tryfona, Theodora, Lenfant, Nicolas, Henrissat, Bernard, Davies, Gideon J., Walton, Paul H., IT University of Copenhagen, University of Cambridge [UK] (CAM), University of York [York, UK], Novozymes, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Biotechnology and Biological Sciences Research Council (grant number BB/L000423) and Agence Française de l'Environnement et de la Maîtrise de l'Energie (grant number 1201C102). The Danish Council for Strategic Research (grant numbers 12-134923 and 12-134922). The Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT and the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570), Dupree, Paul [0000-0001-9270-6286], Tryfona, Theodora [0000-0002-1618-3521], Apollo - University of Cambridge Repository, Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and IT University of Copenhagen (ITU)
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Protein Conformation ,Oligosaccharides ,beta-Amylase ,Crystallography, X-Ray ,Article ,Mixed Function Oxygenases ,Substrate Specificity ,Evolution, Molecular ,Polysaccharides ,Catalytic Domain ,Histidine ,Cellulose ,Maltose ,Phylogeny ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Electron Spin Resonance Spectroscopy ,Fungi ,Starch ,Genomics ,[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,Protein Structure, Tertiary ,Oxygen ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Structural biology ,Acids ,Copper ,Biotechnology - Abstract
Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes., Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes that oxidatively deconstruct polysaccharides. Here, Lo Leggio et al. report the activity, spectroscopy and three-dimensional structure of a LPMO of the new CAZy AA13 family active on recalcitrant-retrograded starch.
- Published
- 2015
15. 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.
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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 kcat/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. A 2,5B 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
16. Molecular Mechanism by which Prominent Human Gut Bacteroidetes Utilize Mixed-Linkage Beta-Glucans, Major Health-Promoting Cereal Polysaccharides.
- Author
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Tamura, Kazune, Hemsworth, Glyn R., Déjean, Guillaume, Rogers, Theresa E., Pudlo, Nicholas A., Urs, Karthik, Jain, Namrata, Davies, Gideon J., Martens, Eric C., and Brumer, Harry
- Abstract
Summary Microbial utilization of complex polysaccharides is a major driving force in shaping the composition of the human gut microbiota. There is a growing appreciation that finely tuned polysaccharide utilization loci enable ubiquitous gut Bacteroidetes to thrive on the plethora of complex polysaccharides that constitute “dietary fiber.” Mixed-linkage β(1,3)/β(1,4)-glucans (MLGs) are a key family of plant cell wall polysaccharides with recognized health benefits but whose mechanism of utilization has remained unclear. Here, we provide molecular insight into the function of an archetypal MLG utilization locus (MLGUL) through a combination of biochemistry, enzymology, structural biology, and microbiology. Comparative genomics coupled with growth studies demonstrated further that syntenic MLGULs serve as genetic markers for MLG catabolism across commensal gut bacteria. In turn, we surveyed human gut metagenomes to reveal that MLGULs are ubiquitous in human populations globally, which underscores the importance of gut microbial metabolism of MLG as a common cereal polysaccharide. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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17. Activity, stability and 3-D structure of the Cu(ii) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens.
- Author
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Gregory, Rebecca C., Hemsworth, Glyn R., Turkenburg, Johan P., Hart, Samuel J., Walton, Paul H., and Davies, Gideon J.
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BIOMASS , *BIOMASS energy , *POLYSACCHARIDES , *GLYCOSIDASES , *GLYCOSIDE synthesis - Abstract
The enzymatic deconstruction of recalcitrant polysaccharide biomass is central to the conversion of these substrates for societal benefit, such as in biofuels. Traditional models for enzyme-catalysed polysaccharide degradation involved the synergistic action of endo-, exo- and processive glycoside hydrolases working in concert to hydrolyse the substrate. More recently this model has been succeeded by one featuring a newly discovered class of mononuclear copper enzymes: lytic polysaccharide monooxygenases (LPMOs; classified as Auxiliary Activity (AA) enzymes in the CAZy classification). In 2013, the structure of an LPMO from Bacillus amyloliquefaciens, BaAA10, was solved with the Cu centre photoreduced to Cu(i) in the X-ray beam. Here we present the catalytic activity of BaAA10. We show that it is a chitin-active LPMO, active on both α and β chitin, with the Cu(ii) binding with low nM KD, and the substrate greatly increasing the thermal stability of the enzyme. A spiral data collection strategy has been used to facilitate access to the previously unobservable Cu(ii) state of the active centre, revealing a coordination geometry around the copper which is distorted from axial symmetry, consistent with the previous findings from EPR spectroscopy. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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- View/download PDF
18. Learning from microbial strategies for polysaccharide degradation.
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Hemsworth, Glyn R., Déjean, Guillaume, Davies, Gideon J., and Brumer, Harry
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POLYSACCHARIDES , *RENEWABLE energy sources , *ENZYME analysis , *BIOTECHNOLOGY , *MONOOXYGENASES - Abstract
Complex carbohydrates are ubiquitous in all kingdoms of life. As major components of the plant cell wall they constitute both a rich renewable carbon source for biotechnological transformation into fuels, chemicals and materials, and also form an important energy source as part of a healthy human diet. In both contexts, there has been significant, sustained interest in understanding how microbes transform these substrates. Classical perspectives of microbial polysaccharide degradation are currently being augmented by recent advances in the discovery of lytic polysaccharide monooxygenases (LPMOs) and polysaccharide utilization loci (PULs). Fundamental discoveries in carbohydrate enzymology are both advancing biological understanding, as well as informing applications in industrial biomass conversion and modulation of the human gut microbiota to mediate health benefits. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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19. Lytic Polysaccharide Monooxygenases in Biomass Conversion.
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Hemsworth, Glyn R., Johnston, Esther M., Davies, Gideon J., and Walton, Paul H.
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MONOOXYGENASES , *BIOMASS conversion , *BIOMASS energy , *LIGNOCELLULOSE , *BIOCHEMISTRY - Abstract
The derivation of second-generation biofuels from non-edible biomass is viewed as crucial for establishing a sustainable bio-based economy for the future. The inertness of lignocellulosic biomass makes its breakdown for conversion into fuels and other compounds a challenge. Enzyme cocktails can be utilized in the bio-refinery for lignocellulose deconstruction but until recently their costs were regarded as high. Lytic polysaccharide monooxygenases (LPMOs) offer tremendous promise for further process improvements owing to their ability to boost the activity of biomass-degrading enzyme consortia. Combining data from multiple disciplines, progress has been made in understanding the biochemistry of LPMOs. We review the academic literature in this area and highlight some of the key questions that remain. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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20. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.
- Author
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Kjaergaard, Christian H., Qayyum, Munzarin F., Wong, Shaun D., Feng Xu, Hemsworth, Glyn R., Walton, Daniel J., Young, Nigel A., Davies, Gideon J., Walton, Paul H., Johansen, Katja Salomon, Hodgson, Keith O., Hedman, Britt, and Solomon, Edward I.
- Subjects
MONOOXYGENASES ,CRYSTAL structure ,X-ray absorption near edge structure ,ABSORPTION spectra ,OXIDATION-reduction reaction - Abstract
Strategies for O
2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(ll) to three-coordinate Cu(l) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(l) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(l) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(ll) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(l) site, which provides an open coordination position for strong O2 binding with very little reorganization energy. [ABSTRACT FROM AUTHOR]- Published
- 2014
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21. Discovery and characterization of a new family of lytic polysaccharide monooxygenases.
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Hemsworth, Glyn R, Henrissat, Bernard, Davies, Gideon J, and Walton, Paul H
- Subjects
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LYSINS , *POLYSACCHARIDES , *MONOOXYGENASES , *BIOMASS energy , *THREE-dimensional imaging , *MOLECULAR structure , *KOJI , *CHITIN - Abstract
Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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- View/download PDF
22. On the catalytic mechanism of dimeric dUTPases.
- Author
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HEMSWORTH, Glyn R., GONZÁLEZ-PACANOWSKA, Dolores, and WILSON, Keith S.
- Subjects
- *
TRYPANOSOMA brucei , *TRYPANOSOMA cruzi , *LEISHMANIA donovani , *PHOSPHATASES , *NUCLEOTIDES ,DEVELOPING countries - Abstract
The Tritryps Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani are responsible for great morbidity and mortality in developing countries. Their dimeric dUTPases are members of the all-α NTP pyrophosphohydrolase family and represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared with the trimeric human enzyme. In the present paper we describe the structure of the T. brucei enzyme in open and closed conformations. Furthermore, we probe thereaction mechanism through the binding of transition state mimics both in solution and inthe crystal. 31P-NMR and tryptophan fluorescence quenching in the presence of AlF3 and MgF3- identified which phosphate is subject to nucleophilic attack by a water molecule. The structures in complex with two transition state analogues confirm that the nucleophilic attack occurs on the β-phosphate in contrast with the α-phosphate in the trimeric enzymes. These results establish the structural basis of catalysis of these important housekeeping enzymes and has ramifications for the wider all-α NTP pyrophosphohydrolase family. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
23. Recent insights into copper-containing lytic polysaccharide mono-oxygenases.
- Author
-
Hemsworth, Glyn R, Davies, Gideon J, and Walton, Paul H
- Subjects
- *
POLYSACCHARIDES , *OXYGENASES , *COPPER , *LYSINS , *BINDING sites , *HISTIDINE , *BIOMASS energy - Abstract
Highlights: [•] Recently discovered polysaccharide mono-oxygenases are copper-containing enzymes. [•] Different sequence families in bacteria and fungi are structurally similar but with subtle differences in active site and mechanism. [•] Copper active sites use N-terminal histidine to coordinate to copper in the so-called ‘histidine brace’. [•] Aided by these enzymes, recalcitrant biomass is becoming a viable biofuel source. [Copyright &y& Elsevier]
- Published
- 2013
- Full Text
- View/download PDF
24. The Copper Active Site of CBM33 Polysaccharide Oxygenases.
- Author
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Hemsworth, Glyn R., Taylor, Edward J., Kim, Robbert Q., Gregory, Rebecca C., Lewis, Sally J., Turkenburg, Johan P., Parkin, Alison, Davies, Gideon J., and Walton, Paul H.
- Subjects
- *
BINDING sites , *COPPER , *POLYSACCHARIDES , *OXYGENASES , *CELLULOSE , *METALLOENZYMES , *X-ray absorption , *HISTIDINE - Abstract
The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual t-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
25. Structure of the Human Obesity Receptor Leptin-Binding Domain Reveals the Mechanism of Leptin Antagonism by a Monoclonal Antibody
- Author
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Carpenter, Byron, Hemsworth, Glyn R., Wu, Zida, Maamra, Mabrouka, Strasburger, Christian J., Ross, Richard J., and Artymiuk, Peter J.
- Subjects
- *
OBESITY treatment , *LEPTIN receptors , *MONOCLONAL antibodies , *CRYSTAL structure , *HOMEOSTASIS , *IMMUNE system , *TARGETED drug delivery - Abstract
Summary: Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin''s mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
26. The Crystal Structure of the Leishmania major Deoxyuridin Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine.
- Author
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Hemsworth, Glyn R., Moroz, Olga V., Fogg, Mark J., Scott, Benjamin, Bosch-Navarrete, Cristina, González-Pacanowska, Dolores, and Wilson, Keith S.
- Subjects
- *
LEISHMANIASIS , *LEISHMANIA , *KINETOPLASTIDA , *CAMPYLOBACTER jejuni , *DRUG development - Abstract
Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
27. Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31.
- Author
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Larsbrink, Johan, Izumi, Atsushi, Hemsworth, Glyn R., Davies, Gideon J., and Brumer, Harry
- Subjects
- *
ENZYMOLOGY , *ENZYME activation , *GLYCOSYLTRANSFERASE genetics , *GLYCOSIDASES , *ENZYME inhibitors , *ENZYME analysis - Abstract
The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
28. Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases.
- Author
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Frandsen, Kristian E. H., Tovborg, Morten, Jørgensen, Christian I., Spodsberg, Nikolaj, Rosso, Marie-Noëlle, Hemsworth, Glyn R., Garman, Elspeth F., Grime, Geoffrey W., Poulsen, Jens-Christian N., Batth, Tanveer S., Shingo Miyauchi, Lipzen, Anna, Daum, Chris, Grigoriev, Igor V., Johansen, Katja S., Henrissat, Bernard, Berrin, Jean-Guy, and Lo Leggio, Leila
- Subjects
- *
MONOOXYGENASES , *FUNGAL enzymes , *HISTIDINE , *WOOD decay , *AMINO acid sequence , *COPPER ions , *POTENTIAL functions - Abstract
Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, is AA9B, a naturally occurring protein from Lentinus similis. The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
29. Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases.
- Author
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Chaplin, Amanda K., Wilson, Michael T., Hough, Michael A., Svistunenko, Dimitri A., Hemsworth, Glyn R., Walton, Paul H., Vijgenboom, Erik, and Worrall, Jonathan A. R.
- Subjects
- *
HISTIDINE , *POLYSACCHARIDES , *MONOOXYGENASES , *AMINO group , *THERMODYNAMICS - Abstract
Copper-dependent lytic polysaccharide monooxygenases (LPMOs)are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10EExt). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
30. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases
- Author
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Esther M. Johnston, Gideon J. Davies, Laurence Marmuse, Morten Tovborg, Leila Lo Leggio, Bernard Henrissat, Katja Salomon Johansen, Floriana Tuna, Amgalanbaatar Baldansuren, Paul H. Walton, Luisa Ciano, Hugues Driguez, Pernille von Freiesleben, Sébastien Fort, Glyn R. Hemsworth, Jens-Christian N. Poulsen, Thomas J. Simmons, Paul Dupree, Sylvain Cottaz, Kristian E. H. Frandsen, Nicolas Lenfant, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), UK Biotechnology and Biological Sciences Research Council [BB/L000423, BB/L021633/1], Agence Francaise de l'Environnement et de la Maitrise de l'Energie [1201C102], Danish Council for Strategic Research [12-134923, 12-134922], European Community [283570], Institut de Chimie Moleculaire de Grenoble [FR 2607], LabEx ARCANE [ANR-11-LABX-0003-01], PolyNat Carnot Institute, French Agence Nationale de la Recherche [PNRB2005-11], Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Dupree, Paul [0000-0001-9270-6286], Hemsworth, Glyn R [0000-0002-8226-1380], Ciano, Luisa [0000-0002-1667-0856], Baldansuren, Amgalanbaatar [0000-0001-8767-3731], Davies, Gideon J [0000-0002-7343-776X], Lo Leggio, Leila [0000-0002-5135-0882], Walton, Paul H [0000-0002-1152-1480], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Models, Molecular ,conformation ,coordination ,oxygenases ,Aspergillus oryzae ,Oligosaccharides ,Chitin ,Lentinula ,Crystallography, X-Ray ,Mixed Function Oxygenases ,Substrate Specificity ,chemistry.chemical_compound ,Catalytic Domain ,Fluorescence Resonance Energy Transfer ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Biochemistry ,Oxidation-Reduction ,Molecular Sequence Data ,mechanism ,Polysaccharide ,chemistry ,Article ,03 medical and health sciences ,cellulose degradation ,Oxidoreductase ,Amino Acid Sequence ,Cellulose ,Molecular Biology ,Binding Sites ,Active site ,Cell Biology ,Monooxygenase ,enzyme ,030104 developmental biology ,Enzyme ,substrate-specificity ,biology.protein ,activation ,Copper ,discovery - Abstract
Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here the first structural determination of an LPMO–oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.
- Published
- 2016
31. Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.
- Author
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Rajagopal BS, Yates N, Smith J, Paradisi A, Tétard-Jones C, Willats WGT, Marcus S, Knox JP, Firdaus-Raih M, Henrissat B, Davies GJ, Walton PH, Parkin A, and Hemsworth GR
- Subjects
- Oxidation-Reduction, Mixed Function Oxygenases, Polysaccharides, Gammaproteobacteria
- Abstract
The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria., (open access.)
- Published
- 2024
- Full Text
- View/download PDF
32. Selectivity and stability of N-terminal targeting protein modification chemistries.
- Author
-
Barber LJ, Yates NDJ, Fascione MA, Parkin A, Hemsworth GR, Genever PG, and Spicer CD
- Abstract
Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no 'one size fits all' approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field., Competing Interests: The authors declare no competing financial interests., (This journal is © The Royal Society of Chemistry.)
- Published
- 2022
- Full Text
- View/download PDF
33. A Standalone β-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold.
- Author
-
Fazal A, Hemsworth GR, Webb ME, and Seipke RF
- Subjects
- Alcohol Oxidoreductases chemistry, Alcohol Oxidoreductases genetics, Amino Acid Sequence, Antimycin A biosynthesis, Antimycin A metabolism, Biocatalysis, Catalytic Domain, Computational Biology, Crystallography, X-Ray, Molecular Docking Simulation, Mutation, Protein Binding, Sequence Alignment, Streptomyces enzymology, Substrate Specificity genetics, Alcohol Oxidoreductases metabolism, Antimycin A analogs & derivatives
- Abstract
Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone β-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone β-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone β-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.
- Published
- 2021
- Full Text
- View/download PDF
34. Insights from semi-oriented EPR spectroscopy studies into the interaction of lytic polysaccharide monooxygenases with cellulose.
- Author
-
Ciano L, Paradisi A, Hemsworth GR, Tovborg M, Davies GJ, and Walton PH
- Subjects
- Apium chemistry, Cellulose metabolism, Electron Spin Resonance Spectroscopy, Magnetic Fields, Mixed Function Oxygenases metabolism, Polysaccharides metabolism, Cellulose chemistry, Mixed Function Oxygenases chemistry, Polysaccharides chemistry
- Abstract
Probing the detailed interaction between lytic polysaccharide monooxygenases (LPMOs) and their polysaccharide substrates is key to revealing further insights into the mechanism of action of this class of enzymes on recalcitrant biomass. This investigation is somewhat hindered, however, by the insoluble nature of the substrates, which precludes the use of most optical spectroscopic techniques. Herein, we report a new semi-oriented EPR method which evaluates directly the binding of cellulose-active LPMOs to crystalline cellulose. We make use of the intrinsic order of cellulose fibres in Apium graveolens (celery) to orient the LPMO with respect to the magnetic field of an EPR spectrometer. The subsequent angle-dependent changes observed in the EPR spectra can then be related to the orientation of the g matrix principal directions with respect to the magnetic field of the spectrometer and, hence, to the binding of the enzyme onto the cellulose fibres. This method, which does not require specific modification of standard CW-EPR equipment, can be used as a general procedure to investigate LPMO-cellulose interactions.
- Published
- 2020
- Full Text
- View/download PDF
35. Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.
- Author
-
Drulyte I, Obajdin J, Trinh CH, Kalverda AP, van der Kamp MW, Hemsworth GR, and Berry A
- Abstract
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of 'tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels-Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99-107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+β barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [
15 N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels-Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels-Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399., (© Ieva Drulyte et al. 2019.)- Published
- 2019
- Full Text
- View/download PDF
36. A Cell-Surface GH9 Endo-Glucanase Coordinates with Surface Glycan-Binding Proteins to Mediate Xyloglucan Uptake in the Gut Symbiont Bacteroides ovatus.
- Author
-
Foley MH, Déjean G, Hemsworth GR, Davies GJ, Brumer H, and Koropatkin NM
- Subjects
- Biological Transport physiology, Cell Membrane metabolism, Cellulase metabolism, Dietary Fiber metabolism, Gastrointestinal Tract metabolism, Glycoside Hydrolases metabolism, Humans, Bacterial Proteins metabolism, Bacteroides metabolism, Gastrointestinal Microbiome physiology, Gastrointestinal Tract microbiology, Glucans metabolism, Membrane Proteins metabolism, Polysaccharides metabolism, Xylans metabolism
- Abstract
Dietary fiber is an important food source for members of the human gut microbiome. Members of the dominant Bacteroidetes phylum capture diverse polysaccharides via the action of multiple cell surface proteins encoded within polysaccharide utilization loci (PUL). The independent activities of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) for the harvest of various glycans have been studied in detail, but how these proteins work together to coordinate uptake is poorly understood. Here, we combine genetic and biochemical approaches to discern the interplay between the BoGH9 endoglucanase and the xyloglucan-binding proteins SGBP-A and SGBP-B from the Bacteroides ovatus xyloglucan utilization locus (XyGUL). The expression of BoGH9, a weakly active xyloglucanase in isolation, is required in a strain that expresses a non-binding version of SGBP-A (SGBP-A*). The crystal structure of the BoGH9 enzyme suggests the molecular basis for its robust activity on mixed-linkage β-glucan compared to xyloglucan. However, catalytically inactive site-directed mutants of BoGH9 fail to complement the deletion of the active BoGH9 in a SGBP-A* strain. We also find that SGBP-B is needed in an SGBP-A* background to support growth on xyloglucan, but that the non-binding SGBP-B* protein acts in a dominant negative manner to inhibit growth on xyloglucan. We postulate a model whereby the SGBP-A, SGBP-B, and BoGH9 work together at the cell surface, likely within a discrete complex, and that xyloglucan binding by SGBP-B and BoGH9 may facilitate the orientation of the xyloglucan for transfer across the outer membrane., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
37. Production and spectroscopic characterization of lytic polysaccharide monooxygenases.
- Author
-
Hemsworth GR, Ciano L, Davies GJ, and Walton PH
- Subjects
- Electron Spin Resonance Spectroscopy, Escherichia coli genetics, Escherichia coli metabolism, Mixed Function Oxygenases genetics, Recombinant Proteins genetics, Substrate Specificity, Mixed Function Oxygenases metabolism, Recombinant Proteins metabolism
- Abstract
Lytic polysaccharide monooxygenases (LPMOs, also known as PMOs) are a recently discovered family of enzymes that play a key role in the breakdown of polysaccharide substrates. The ability of LPMOs to introduce chain breaks, using an oxidative mechanism, has particularly attracted attention as the world seeks more cost-effective and environmentally friendly ways of producing second-generation biofuels for the future. LPMOs are copper-dependent enzymes and have an unusual active site which includes the N-terminal residue of the protein in the copper coordination sphere. This N-terminal histidine side chain is also methylated in fungal enzymes, the molecular reason for which is still a debated topic. The production of these enzymes poses several challenges if we are to understand their chemical mechanisms. Here, we describe the methods that have been used in the field to produce LPMOs and provide information on the workflows that we use for our electron paramagnetic resonance (EPR) spectroscopy experiments. EPR has been a particularly powerful tool in the study of these enzymes and our objective with this chapter is to provide some helpful information for researchers for whom this technique might be daunting or theoretically difficult to access., (© 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
38. Structural and functional insight into human O-GlcNAcase.
- Author
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Roth C, Chan S, Offen WA, Hemsworth GR, Willems LI, King DT, Varghese V, Britton R, Vocadlo DJ, and Davies GJ
- Subjects
- Acetylglucosamine metabolism, Binding Sites, Enzyme Activation drug effects, Enzyme Inhibitors pharmacology, HEK293 Cells, Humans, Ligands, Protein Binding, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Structure, Tertiary, beta-N-Acetylhexosaminidases genetics, beta-N-Acetylhexosaminidases metabolism, Models, Molecular, beta-N-Acetylhexosaminidases chemistry
- Abstract
O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value.
- Published
- 2017
- Full Text
- View/download PDF
39. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.
- Author
-
Frandsen KE, Simmons TJ, Dupree P, Poulsen JC, Hemsworth GR, Ciano L, Johnston EM, Tovborg M, Johansen KS, von Freiesleben P, Marmuse L, Fort S, Cottaz S, Driguez H, Henrissat B, Lenfant N, Tuna F, Baldansuren A, Davies GJ, Lo Leggio L, and Walton PH
- Subjects
- Amino Acid Sequence, Aspergillus oryzae enzymology, Aspergillus oryzae genetics, Binding Sites, Catalytic Domain, Copper metabolism, Crystallography, X-Ray, Fluorescence Resonance Energy Transfer, Lentinula enzymology, Lentinula genetics, Mixed Function Oxygenases chemistry, Mixed Function Oxygenases genetics, Models, Molecular, Molecular Sequence Data, Oligosaccharides chemistry, Oxidation-Reduction, Substrate Specificity, Cellulose metabolism, Chitin metabolism, Mixed Function Oxygenases metabolism
- Abstract
Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.
- Published
- 2016
- Full Text
- View/download PDF
40. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.
- Author
-
Kjaergaard CH, Qayyum MF, Wong SD, Xu F, Hemsworth GR, Walton DJ, Young NA, Davies GJ, Walton PH, Johansen KS, Hodgson KO, Hedman B, and Solomon EI
- Subjects
- Catalysis, Catalytic Domain, Chitin chemistry, Computer Simulation, Electron Spin Resonance Spectroscopy, Electrons, Models, Molecular, Spectrophotometry, Superoxides chemistry, Thermodynamics, X-Rays, Copper chemistry, Fungal Proteins chemistry, Mixed Function Oxygenases chemistry, Oxygen chemistry, Polysaccharides chemistry, Thermoascus enzymology
- Abstract
Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.
- Published
- 2014
- Full Text
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