| 1. |
- Bi, Ran, 1985-
(författare)
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Lignocellulose Degradation by Soil Micro-organisms
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic biomass is a sustainable resource with abundant reserves. Compared to petroleum ‐ based products, the biomass ‐ derived polymers and chemicals give better environmental profiles. A lot of research interest is focused on understanding the lignocellulose structures.Lignin, among the three major wood components, represents most difficulty for microbial degradation because of its complex structure and because cross ‐ linking to hemicellulose makes wood such a compact structure. Nevertheless, wood is naturally degraded by wood ‐ degrading micro ‐ organisms and modified and partly degraded residual of lignin goes into soil. Therefore soil serves as a good environment in which to search for special lignin ‐ degraders. In this thesis, different types of lignin have been used as sole carbon sources to screen for lignin ‐ degrading soil micro ‐ organisms. Eleven aerobic and three anaerobic microbe strains have been isolated and identified as able to grow on lignin. The lignin degradation patterns of selected strains have been studied and these partly include an endwise cleavage of β‐ O ‐ 4 bonds in lignin and is more complex than simple hydrolytic degradation.As lignin exists in wood covalently bonded to hemicellulose, one isolated microbe strain, Phoma herbarum, has also been studied with regards to its ability to degrade covalent lignin polysaccharide networks (LCC). The results show that its culture filtrate can attack lignin ‐ polysaccharide networks in a manner different from that of the commercial enzyme product, Gammanase, possibly by selective cleavage of phenyl glucoside bonds. The effects on LCC of Phoma herbarum also enhance polymer extractability. Hot ‐ water extraction of a culture filtrate of Phoma herbarum ‐ treated fiberized spruce wood material gave an amount of extracted galactoglucomannan more than that given by the Gammanase ‐ treated material and non ‐ enzyme ‐ treated material.Over millions of years of natural evolution, micro ‐ organisms on the one hand develop so that they can degrade all wood components to get energy for growth, while plants on the other hand also continuously develop to defend from microbial attack. Compared with lignin and cellulose, hemicelluloses as major components of plant cell walls, are much more easily degraded, but hemicelluloses differ from cellulose in that they are acetylated to different extents. The biological functions of acetylation are not completely understood, but it is suggested is that one function is to decrease the microbial degradability of cell walls. By cultivation of soil micro ‐ organisms using mannans acetylated to deffernent degrees as sole carbon source on agar plates, we were able to see significant trends where the resistance towards microbial degradation of glucomannan and galactomannan increased with increasing degree of acetylation. Possible mechanisms and the technological significance of this are discussed. Tailoring the degree of acetylation of polysaccharide materials might slow down the biodegradation, making it possible to design a material with a degradation rate suited to its application.
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| 2. |
- Blomqvist, Johanna, et al.
(författare)
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Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis
- 2011
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Ingår i: Letters in Applied Microbiology. - Malden, USA : Wiley-Blackwell. - 0266-8254 .- 1472-765X. ; 53:1, s. 73-78
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Tidskriftsartikel (refereegranskat)abstract
- Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae.Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small-scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l(-1) (0.22 mol l(-1))]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0 42 +/- 0 01 g ethanol (g glucose)(-1) were observed for both yeasts in 1 : 10 hydrolysate. In small-scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate.Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate.Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.
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| 3. |
- Borisova, Anna, et al.
(författare)
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Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, similar to 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs.Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.
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| 4. |
- Borisova, Anna, et al.
(författare)
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Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C
- 2015
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Ingår i: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 282, s. 4515-4537
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Tidskriftsartikel (refereegranskat)abstract
- The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 degrees C versus 60-65 degrees C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported - on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A.
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| 5. |
- Dererie, Debebe Yilma, et al.
(författare)
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Improved bio-energy yields via sequential ethanol fermentation and biogas digestion of steam exploded oat straw
- 2011
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Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 102:6, s. 4449-4455
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Tidskriftsartikel (refereegranskat)abstract
- Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85-87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5-9.8 MJ/(kg dry oat straw), which is 28-34% higher than direct biogas digestion that yielded 7.3-7.4 MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24 days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process.
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| 6. |
- Fagerström, Alexandra, et al.
(författare)
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Chiral recognition mechanism of cellobiohydrolase Cel7A for ligands based on the β‐blocker propranolol : The effect of explicit water molecules on binding and selectivities
- 2023
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Ingår i: Natural Sciences. - : John Wiley & Sons. - 2698-6248 .- 2698-6248. ; 3:3
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Tidskriftsartikel (refereegranskat)abstract
- Proteins are useful chiral selectors. In order to understand the recognition mechanism and chiral discrimination, the binding of the (R)- and (S)-enantiomers of a series of designed amino alcohol inhibitors based on propranolol to cellobiohydrolase Cel7A (Trichoderma reesei) has been studied more closely. X-ray crystal structures were determined of the protein complex with the (R)- and (S)-enantiomers of the strongest binding propranolol analogue. The combination of the structural data, thermodynamic data from capillary electrophoresis and microcalorimetry experiments and computational modelling give a clearer insight into the origin of the enantioselectivity and its opposite thermodynamic signature. The new crystal structures were used in computational molecular flexible dockings of the propranolol analogues using the program Glide. The results indicated that several water molecules in the active site were essential for the docking of the (R)-enantiomers but not for the (S)-enantiomers. The results are discussed in relation to the enantiomeric discrimination of the enzyme. Both dissociation constants (Kd values) and thermodynamical data are included to show the effects of the structural modifications in the ligand on enthalpy and entropy in relation to the enantioselectivity.
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| 7. |
- Gudmundsson, Mikael, et al.
(författare)
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Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction
- 2014
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289, s. 18782-18792
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Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.
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| 8. |
- Gudmundsson, Mikael, et al.
(författare)
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The dissociation mechanism of processive cellulases
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116, s. 23061-23067
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Tidskriftsartikel (refereegranskat)abstract
- Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.
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| 9. |
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| 10. |
- Gullfot, Fredrika, 1967-
(författare)
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Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.
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| 11. |
- Haataja, Topi, et al.
(författare)
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Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding : insights from crystal structures and MD simulation
- 2023
-
Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 290:2, s. 379-399
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Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-beta-d-cellobioside (pNPC), p-nitrophenyl-beta-d-lactoside (pNPL) and methylumbelliferyl-beta-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-beta-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (k(cat)) than TrCel7A, but higher Michaelis-Menten (K-M) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.
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| 12. |
- Haataja, Topi, et al.
(författare)
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The crystal structure of RsSymEG1 reveals a unique form of smaller GH7 endoglucanases alongside GH7 cellobiohydrolases in protist symbionts of termites
- 2024
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Ingår i: The Febs Journal. - 1742-464X .- 1742-4658. ; 291, s. 1168-1185
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo-acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60-80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non-reducing end of the cellulose-binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms.
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| 13. |
- Haddad Momeni, Majid, et al.
(författare)
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Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life
- 2016
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 82, s. 3395-3409
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 angstrom and 2.7 angstrom respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A(CBM) and DpuCel7A(CBM), which were recombinantly expressed in T. reesei. DdiCel7A(CBM) and DpuCel7A(CBM) hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K-i of cellobiose was significantly higher for DdiCel7A(CBM) and DpuCel7A(CBM) than for TreCel7A: 205, 130, and 29 mu M, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.IMPORTANCEGH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate.
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| 14. |
- Haddad Momeni, Majid, et al.
(författare)
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Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea
- 2014
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Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 70, s. 2356-2366
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10 degrees C higher melting temperature (T-m of 72.5 degrees C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65 degrees C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 angstrom resolution; R-work and R-free of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.
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| 15. |
- Haddad Momeni, Majid, et al.
(författare)
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Improved bio-energy yields via sequential ethanol fermentation and biogas digestion of steam exploded oat straw
- 2011
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 102, s. 4449-4455
-
Tidskriftsartikel (refereegranskat)abstract
- Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85-87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5-9.8 MJ/(kg dry oat straw), which is 28-34% higher than direct biogas digestion that yielded 7.3-7.4 MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24 days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process. (C) 2010 Elsevier Ltd. All rights reserved.
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| 16. |
- Haddad Momeni, Majid, et al.
(författare)
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Structural, Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:8, s. 5861-5872
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Tidskriftsartikel (refereegranskat)abstract
- Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 angstrom resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the -7 to -4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo- initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.
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| 17. |
- Haddad Momeni, Majid, et al.
(författare)
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Structural insights into the inhibition of cellobiohydrolase Cel7A by xylo-oligosaccharides
- 2015
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 282:11, s. 2167-2177
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Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Hypocreajecorina (anamorph of Trichodermareesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides (XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H.jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted similar to 2.4 angstrom towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition. DatabaseThe atomic coordinates and structure factors are available in the Protein Data Bank under accession number (H. jecorina Cel7A E212Q variant, complex with xylotriose), (H. jecorina Cel7A E217Q variant, complex with xylotriose), (H. jecorina Cel7A E212Q variant, complex with xylopentaose), (H. jecorina Cel7A E217Q variant, complex with xylopentaose), (wild-type H. jecorina Cel7A, complex with xylopentaose) and (H. jecorina Cel7A E217Q variant, complex with xylotetraose).
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| 18. |
- Haddad Momeni, Majid, et al.
(författare)
-
The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies
- 2014
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136, s. 321-329
-
Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.
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| 19. |
- Hamark, Christoffer, et al.
(författare)
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Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A
- 2018
-
Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 24:68, s. 17975-17985
-
Tidskriftsartikel (refereegranskat)abstract
- At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by H-1,H-1 and H-1,C-13 scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.
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| 20. |
- Henriksson, Gunnar, et al.
(författare)
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Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase.
- 1999
-
Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 259:1-2, s. 88-95
-
Tidskriftsartikel (refereegranskat)abstract
- A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.
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| 21. |
- Karkehabadi, Saeid, et al.
(författare)
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Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution
- 2017
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 292, s. 17418-17430
-
Tidskriftsartikel (refereegranskat)abstract
- Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 degrees C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 degrees C increase in T-m and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 degrees C. The X-ray crystal structure of the catalytic domain was determined at 2.1 angstrom resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.
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| 22. |
- Koivula, Anu, et al.
(författare)
-
The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175.
- 2002
-
Ingår i: J Am Chem Soc. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 124:34, s. 10015-24
-
Tidskriftsartikel (refereegranskat)abstract
- Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.
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| 23. |
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| 24. |
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| 25. |
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| 26. |
- Levenfors, Jolanta, et al.
(författare)
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Antibacterial pyrrolidinyl and piperidinyl substituted 2,4-diacetylphloroglucinols from Pseudomonas protegens UP46
- 2020
-
Ingår i: Journal of antibiotics (Tokyo. 1968). - : SPRINGERNATURE. - 0021-8820 .- 1881-1469. ; 73:11, s. 739-747
-
Tidskriftsartikel (refereegranskat)abstract
- In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG (1) and 6-(piperidin-2-yl)DAPG (2). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with minimal inhibitory concentration (MIC) 2 and 4 mu g ml(-1), respectively, for 1, and 2 mu g ml(-1) for both pathogens for 2. The MICs for 1 and 2, against all tested Gram-negative bacteria, were >32 mu g ml(-1). The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 mu g ml(-1), respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2, and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2, but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.
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| 27. |
- Liu, Bing, et al.
(författare)
-
Side-by-side biochemical comparison of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare on their activity against crystalline cellulose and glucomannan
- 2018
-
Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 13:9
-
Tidskriftsartikel (refereegranskat)abstract
- Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLP-MO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood.
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| 28. |
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| 29. |
- Muñoz, Inés G., et al.
(författare)
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Family 7 cellobiohydrolases from Phanerochaete chrysosporium : Crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 Å resolution and homology models of the isozymes
- 2001
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 314:5, s. 1097-1111
-
Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporimn, constituting approximately 10% of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase 1 (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 Angstrom resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 Angstrom resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.
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| 30. |
- Nutt, Anu, 1971-
(författare)
-
Hydrolytic and Oxidative Mechanisms Involved in Cellulose Degradation
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, Hypocrea jecorina and Phanerochaete chrysosporium, including both the action of the individual enzymes and their synergistic interplay. The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases.The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface.A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases.Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation.Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.
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| 31. |
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| 32. |
- Okmane, Laura, et al.
(författare)
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Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A : Crystal structure and activity comparison with GH45 subfamily A, B and C
- 2022
-
Ingår i: Carbohydrate Polymers. - : Elsevier. - 0144-8617 .- 1879-1344. ; 277
-
Tidskriftsartikel (refereegranskat)abstract
- The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a beta-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported.Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 degrees C relative to 30 degrees C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.
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| 33. |
- Okmane, Laura, et al.
(författare)
-
The first crystal structure of a family 45 glycoside hydrolase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A
- 2024
-
Ingår i: FEBS open bio. - 2211-5463. ; 14, s. 505-514
-
Tidskriftsartikel (refereegranskat)abstract
- Here we describe the first crystal structure of a beta-1,4-endoglucanase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A, which belongs to subfamily C of glycoside hydrolase family 45 (GH45). GtCel45A is similar to 18 kDa in size and the crystal structure contains 179 amino acids. The structure is refined at 1.30 angstrom resolution and R-free 0.18. The enzyme consists of a single catalytic module folded into a six-stranded double-psi beta-barrel domain surrounded by long loops. GtCel45A is very similar in sequence (82% identity) and structure to PcCel45A from the white-rot fungus Phanerochaete chrysosporium. Surprisingly though, initial hydrolysis of barley beta-glucan was almost twice as fast in GtCel45A as compared to PcCel45A.
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| 34. |
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| 35. |
- Passoth, Volkmar, et al.
(författare)
-
Airtight storage of moist wheat grain improves bioethanol yields
- 2009
-
Ingår i: Biotechnology for Biofuels. - London, United Kingdom : Springer Science and Business Media LLC. - 1754-6834. ; 2
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Drying is currently the most frequently used conservation method for cereal grain, which in temperate climates consumes a major part of process energy. Airtight storage of moist feed grain using the biocontrol yeast Pichia anomala as biopreservation agent can substantially reduce the process energy for grain storage. In this study we tested the potential of moist stored grain for bioethanol production.Results: The ethanol yield from moist wheat was enhanced by 14% compared with the control obtained from traditionally (dry) stored grain. This enhancement was observed independently of whether or not P. anomala was added to the storage system, indicating that P. anomala does not impair ethanol fermentation. Starch and sugar analyses showed that during pre-treatment the starch of moist grain was better degraded by amylase treatment than that of the dry grain. Additional pre-treatment with cellulose and hemicellulose-degrading enzymes did not further increase the total ethanol yield. Sugar analysis after this pre-treatment showed an increased release of sugars not fermentable by Saccharomyces cerevisiae.Conclusion: The ethanol yield from wheat grain is increased by airtight storage of moist grain, which in addition can save substantial amounts of energy used for drying the grain. This provides a new opportunity to increase the sustainability of bioethanol production.
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| 36. |
- Passoth, Volkmar, et al.
(författare)
-
Enhanced ethanol production from wheat straw by integrated storage and pre-treatment (ISP)
- 2013
-
Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 52, s. 105-110
-
Tidskriftsartikel (refereegranskat)abstract
- Integrated storage and pre-treatment (ISP) combines biopreservation of moist material under airtight conditions and pre-treatment. Moist wheat straw was inoculated with the biocontrol yeast Wickerhamomyces anomalus, the xylan degrading yeast Scheffersomyces stipitis or a co-culture of both. The samples and non-inoculated controls were stored at 4 or 15 degrees C. The non-inoculated controls were heavily contaminated with moulds, in contrast to the samples inoculated with W. anomalus or S. stipitis. These two yeasts were able to grow on wheat straw as sole source of nutrients. When ethanol was produced from moist wheat straw stored for four weeks at 4 degrees C with S. stipitis, and up to 40% enhanced yield (final yield 0.15g ethanol per g straw dry weight) was obtained compared to a dry sample (0.107 g/g). In all other moist samples, stored for four weeks at 4 degrees C or 15 degrees C, 6-35% higher yields were obtained. Thus, energy efficient bio-preservation can improve the pre-treatment efficiency for lignocellulose biomass, which is a critical bottleneck in its conversion to biofuels. (C) 2012 Elsevier Inc. All rights reserved.
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| 37. |
- Payne, Christina, et al.
(författare)
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Fungal Cellulases
- 2015
-
Ingår i: Chemical Reviews. - : American Chemical Society (ACS). - 0009-2665 .- 1520-6890. ; 115, s. 1308-1448
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Forskningsöversikt (refereegranskat)
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| 38. |
- Sabljić, Igor, et al.
(författare)
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Expression and purification of the type II metacaspase from a unicellular green alga Chlamydomonas reinhardtii
- 2022
-
Ingår i: Plant proteases and plant cell death. - New York, NY : Humana Press. - 9781071620786 - 9781071620793 ; , s. 13-20
-
Bokkapitel (refereegranskat)abstract
- Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.
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| 39. |
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| 40. |
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| 41. |
- Sandgren, Mats, et al.
(författare)
-
Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose
- 2013
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 110, s. 14646-14651
-
Tidskriftsartikel (refereegranskat)abstract
- Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.
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| 42. |
- Sandgren, Mats, et al.
(författare)
-
Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases
- 2021
-
Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 297
-
Tidskriftsartikel (refereegranskat)abstract
- Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These enzymes are often bimodular, including a catalytic domain and carbohydrate-binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain data-driven insights into relation-ships between sequence, structure, and function across the GH7 family. Machine-learning models, trained only on the number of residues in the active-site loops as features, were able to discriminate GH7 CBHs and EGs with up to 99% ac-curacy, demonstrating that the lengths of loops A4, B2, B3, and B4 strongly correlate with functional subtype across the GH7 family. Classification rules were derived such that specific residues at 42 different sequence positions each predicted the functional subtype with accuracies surpassing 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. Our machine learning results recapitulate, as top-performing features, a substantial number of the sequence positions determined by previous experimental studies to play vital roles in GH7 activity. We surmise that the yet-to-be-explored sequence positions among the top-performing features also contribute to GH7 functional variation and may be exploited to understand and manipulate function.
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| 43. |
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| 44. |
- Sandgren, Mats, et al.
(författare)
-
Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes
- 2018
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 28, s. 3-8
-
Annan publikation (refereegranskat)abstract
- CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.
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| 45. |
- Sandgren, Mats, et al.
(författare)
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The Putative Endoglucanase PcGH61D from Phanerochaete chrysosporium Is a Metal-Dependent Oxidative Enzyme that Cleaves Cellulose
- 2011
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.
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| 46. |
- Sandgren, Mats, et al.
(författare)
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The Structure of a Bacterial Cellobiohydrolase : The Catalytic Core of the Thermobifida fusca Family GH6 Cellobiohydrolase Cel6B
- 2013
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 425:3, s. 622-635
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Tidskriftsartikel (refereegranskat)abstract
- Cellulases, glycoside hydrolases that catalyze the degradation of cellulose, are classified as either endoglucanases or cellobiohydrolases (CBHs) based on their architecture and mode of action on the cellulose. CBHs bind the cellulose chain in a more or less closed tunnel and cleave off cellobiose units processively from one end of the cellulosic polymer, while endoglucanases have their active sites in a more or less open cleft and show a higher tendency to cut bonds internally in the polymer. The CBH Cel6A (also called CBH2) from the ascomycete Hypocrea jecorina has a much shorter substrate-binding tunnel and seems less processive than the CBH Cel7A (CBH1), from the same fungus. Here, we present the X-ray crystal structure of the catalytic domain of the CBH Cel6B, also called E3, from the soil bacterium Thermobifida fusca, both in its apo form and co-crystallized with cellobiose. The enzyme structure reveals that the Cel6B enzyme has a much longer substrate-binding site than its fungal GH6 counterparts. The tunnel is comparable in length to that of GH7 CBHs. In the ligand structure with cellobiose, the tunnel exit is completely closed by a 13-residue loop not present in fungal GH6 enzymes. The loop needs to be displaced to allow cellobiose product release for a processive action by the enzyme. When ligand is absent, seven of these residues are not visible in the electron density and the tunnel exit is open.
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| 47. |
- Stael, Simon, et al.
(författare)
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Structure-function study of a Ca2+-independent metacaspase involved in lateral root emergence
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 120
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Tidskriftsartikel (refereegranskat)abstract
- Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.
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| 48. |
- Ståhlberg, Jerry
(författare)
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Advantages of a distant cellulase catalytic base
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 293, s. 4680-4687
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Tidskriftsartikel (refereegranskat)abstract
- The inverting glycoside hydrolase Trichoderma reesei (Hypocrea jecorina) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle.
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| 49. |
- Ståhlberg, Jerry
(författare)
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Carbohydrate-Protein Interactions That Drive Processive Polysaccharide Translocation in Enzymes Revealed from a Computational Study of Cellobiohydrolase Processivity
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136, s. 8810-8819
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Tidskriftsartikel (refereegranskat)abstract
- Translocation of carbohydrate polymers through protein tunnels and clefts is a ubiquitous biochemical phenomenon in proteins such as polysaccharide synthases, glycoside hydrolases, and carbohydrate-binding modules. Although static snapshots of carbohydrate polymer binding in proteins have long been studied via crystallography and spectroscopy, the molecular details of polysaccharide chain processivity have not been elucidated. Here, we employ simulation to examine how a cellulose chain translocates by a disaccharide unit during the processive cycle of a glycoside hydrolase family 7 cellobiohydrolase. Our results demonstrate that these biologically and industrially important enzymes employ a two-step mechanism for chain threading to form a Michaelis complex and that the free energy barrier to chain threading is significantly lower than the hydrolysis barrier. Taken with previous studies, our findings suggest that the rate-limiting step in enzymatic cellulose degradation is the glycosylation reaction, not chain processivity. Based on the simulations, we find that strong electrostatic interactions with polar residues that are conserved in GH7 cellobiohydrolases, but not in GH7 endoglucanases, at the leading glucosyl ring provide the thermodynamic driving force for polysaccharide chain translocation. Also, we consider the role of aromatic carbohydrate interactions, which are widespread in carbohydrate-active enzymes and have long been associated with processivity. Our analysis suggests that the primary role for these aromatic residues is to provide tunnel shape and guide the carbohydrate chain to the active site. More broadly, this work elucidates the role of common protein motifs found in carbohydrate-active enzymes that synthesize or depolymerize polysaccharides by chain translocation mechanisms coupled to catalysis.
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| 50. |
- Ståhlberg, Jerry
(författare)
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Development of minimal enzyme cocktails for hydrolysis of sulfite-pulped lignocellulosic biomass
- 2017
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 246, s. 16-23
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Tidskriftsartikel (refereegranskat)abstract
- Despite recent progress, saccharification of lignocellulosic biomass is still a major cost driver in biorefining. In this study, we present the development of minimal enzyme cocktails for hydrolysis of Norway spruce and sugarcane bagasse, which were pretreated using the so-called BALI(TM) process, which is based on sulfite pulping technology. Minimal enzyme cocktails were composed using several glycoside hydrolases purified from the industrially relevant filamentous fungus Trichoderma reesei and a purified commercial beta-glucosidase from Aspergillus niger. The contribution of in-house expressed lytic polysaccharide monooxygenases (LPMOs) was also tested, since oxidative cleavage of cellulose by such LPM05 is known to be beneficial for conversion efficiency. We show that the optimized cocktails permit efficient saccharification at reasonable enzyme loadings and that the effect of the LPMOs is substrate-dependent. Using a cocktail comprising only four enzymes, glucan conversion for Norway spruce reached > 80% at enzyme loadings of 8 mg/g glucan, whereas almost 100% conversion was achieved at 16 mg/g. (C) 2017 Elsevier B.V. All rights reserved.
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