Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    We will mail you new results for this query: keywords==archaeon pyrococcus-furiosus
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Virus-like particle nanoreactors: programmed en capsulation of the thermostable CelB glycosidase inside the P22 capsid
Patterson, D.P. ; Schwarz, B. ; El-Boubbou, K. ; Oost, J. van der; Prevelige, P.E. ; Douglas, T. - \ 2012
Soft Matter 8 (2012)39. - ISSN 1744-683X - p. 10158 - 10166.
beta-glucosidase celb - archaeon pyrococcus-furiosus - protein cages - scaffolding protein - bacteriophage p22 - silica immobilization - enzyme immobilization - gold nanoparticles - functional domains - escherichia-coli
Self-assembling biological systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically active nanoreactors. The in vivo assembly and encapsulation produces P22 VLPs with a high packaging density of the tetrameric CelB, but without loss of enzyme activity or the ability of the P22 VLP to undergo unique morphological transitions that modify the VLPs internal volume and shell porosity. The P22 VLPs encapsulating CelB are also shown to retain a high percentage of the enzyme activity upon being embedded and immobilized in a polymeric matrix
Correlated mutation analyses on super-family alignments reveal functionally important residues
Kuipers, R.K. ; Joosten, H.J. ; Verwiel, E. ; Paans, J. ; Akerboom, J. ; Oost, J. van der; Leferink, N.G.H. ; Berkel, W.J.H. van; Vriend, G. ; Schaap, P.J. - \ 2009
Proteins : Structure, Function, and Bioinformatics 76 (2009)3. - ISSN 0887-3585 - p. 608 - 616.
furiosus phosphoglucose isomerase - archaeon pyrococcus-furiosus - substrate-binding - hiv-1 protease - superfamily - proteins - enzyme - determinants - conservation - receptors
Correlated mutation analyses (CMA) on multiple sequence alignments are widely used for the prediction of the function of amino acids. The accuracy of CMA-based predictions is mainly determined by the number of sequences, by their evolutionary distances, and by the quality of the alignments. These criteria are best met in structure-based sequence alignments of large super-families. So far, CMA-techniques have mainly been employed to study the receptor interactions. The present work shows how a novel CMA tool, called Comulator, can be used to determine networks of functionally related residues in enzymes. These analyses provide leads for protein engineering studies that are directed towards modification of enzyme specificity or activity. As proof of concept, Comulator has been applied to four enzyme super-families: the isocitrate lyase/phoshoenol-pyruvate mutase super-family, the hexokinase super-family, the RmlC-like cupin super-family, and the FAD-linked oxidases super-family. In each of those cases networks of functionally related residue positions were discovered that upon mutation influenced enzyme specificity and/or activity as predicted. We conclude that CMA is a powerful tool for redesigning enzyme activity and selectivity
The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner-Doudoroff pathway
Ettema, T.J.G. ; Ahmed, H. ; Geerling, A.C.M. ; Oost, J. van der; Siebers, B. - \ 2008
Extremophiles 12 (2008)1. - ISSN 1431-0651 - p. 75 - 88.
central carbohydrate-metabolism - archaeon pyrococcus-furiosus - dna microarray analysis - thermoproteus-tenax - aldehyde dehydrogenase - hyperthermophilic archaeon - glucose-dehydrogenase - thermoplasma-acidophilum - glycolytic pathways - structural basis
Archaea utilize a branched modification of the classical Entner¿Doudoroff (ED) pathway for sugar degradation. The semi-phosphorylative branch merges at the level of glyceraldehyde 3-phosphate (GAP) with the lower common shunt of the Emden-Meyerhof-Parnas pathway. In Sulfolobus solfataricus two different GAP converting enzymes¿classical phosphorylating GAP dehydrogenase (GAPDH) and the non-phosphorylating GAPDH (GAPN)¿were identified. In Sulfolobales the GAPN encoding gene is found adjacent to the ED gene cluster suggesting a function in the regulation of the semi-phosphorylative ED branch. The biochemical characterization of the recombinant GAPN of S. solfataricus revealed that¿like the well-characterized GAPN from Thermoproteus tenax¿the enzyme of S. solfataricus exhibits allosteric properties. However, both enzymes show some unexpected differences in co-substrate specificity as well as regulatory fine-tuning, which seem to reflect an adaptation to the different lifestyles of both organisms. Phylogenetic analyses and database searches in Archaea indicated a preferred distribution of GAPN (and/or GAP oxidoreductase) in hyperthermophilic Archaea supporting the previously suggested role of GAPN in metabolic thermoadaptation. This work suggests an important role of GAPN in the regulation of carbon degradation via modifications of the EMP and the branched ED pathway in hyperthermophilic Archaea.
Kinetically controlled refolding of a heat denatured hyperthermostable protein
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2007
FEBS Journal 274 (2007)22. - ISSN 1742-464X - p. 5915 - 5923.
archaeon pyrococcus-furiosus - crystal-structure - thermodynamic properties - thermal-denaturation - citrate synthase - stability - temperature - enzyme - water - dehydrogenase
The thermal denaturation of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144¿°C, corresponding to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, previously denatured protein samples gave well-resolved denaturation transition peaks and showed enzymatic activity after 3 and 9¿months of storage, indicating slow refolding to the native conformation over time.
A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes
Kanai, T. ; Akerboom, A.P. ; Takedomi, S. ; Werken, H.J.G. van de; Blombach, F. ; Oost, J. van der; Murakami, T. ; Atomi, H. ; Imanaka, T. - \ 2007
Journal of Biological Chemistry 282 (2007)46. - ISSN 0021-9258 - p. 33659 - 33670.
archaeon pyrococcus-furiosus - embden-meyerhof pathway - adp-dependent phosphofructokinase - central carbohydrate-metabolism - hyperthermophilic archaeon - thermoproteus-tenax - escherichia-coli - glyceraldehyde-3-phosphate dehydrogenase - abc transporter - bindin
We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (tgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the tgr mutant even under gluconeogenic conditions. The tgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.
Hyperthermophilic enzymes - stability, activity and implementation strategies for high temperature applications
Unsworth, L.D. ; Oost, J. van der; Koutsopoulos, S. - \ 2007
FEBS Journal 274 (2007)16. - ISSN 1742-464X - p. 4044 - 4056.
archaeon pyrococcus-furiosus - extremely thermophilic archaebacteria - thermostable dna-polymerase - alpha-glucosidase gene - sulfolobus-solfataricus - escherichia-coli - biochemical-characterization - thermococcus-litoralis - thermotoga-maritima - protein stabili
Current theories agree that there appears to be no unique feature responsible for the remarkable heat stability properties of hyperthermostable proteins. A concerted action of structural, dynamic and other physicochemical attributes are utilized to ensure the delicate balance between stability and functionality of proteins at high temperatures. We have thoroughly screened the literature for hyperthermostable enzymes with optimal temperatures exceeding 100 °C that can potentially be employed in multiple biotechnological and industrial applications and to substitute traditionally used, high-cost engineered mesophilic/thermophilic enzymes that operate at lower temperatures. Furthermore, we discuss general methods of enzyme immobilization and suggest specific strategies to improve thermal stability, activity and durability of hyperthermophilic enzymes.
Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family
Thaw, P. ; Sedelnikova, S.E. ; Muranova, T. ; Wiese, S. ; Ayora, S. ; Alonso, J.C. ; Brinkman, A.B. ; Akerboom, A.P. ; Oost, J. van der; Rafferty, J.B. - \ 2006
Nucleic acids research 34 (2006)5. - ISSN 0305-1048 - p. 1439 - 1449.
bacillus-subtilis lrpc - archaeon pyrococcus-furiosus - escherichia-coli lrp - sulfolobus-solfataricus - dna-binding - crystal-structure - agrobacterium-tumefaciens - macromolecular structures - global regulator - protein
The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator¿effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.
Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily
Machielsen, M.P. ; Uria, A.R. ; Kengen, S.W.M. ; Oost, J. van der - \ 2006
Applied and Environmental Microbiology 72 (2006)1. - ISSN 0099-2240 - p. 233 - 238.
archaeon pyrococcus-furiosus - aldehyde ferredoxin oxidoreductase - tungsten-containing enzyme - thermococcus strain es-1 - crystal-structure - molecular characterization - escherichia-coli - proteins - binding - sulfur
The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.
Structural features of a hyperthermostable endo-beta-1,3-glucanase in solution and adsorbed on 'invisible' particles
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2005
Biophysical Journal 88 (2005)1. - ISSN 0006-3495 - p. 467 - 474.
archaeon pyrococcus-furiosus - time-resolved fluorescence - circular-dichroism - proteins - adsorption - peroxidase - interfaces - stability - mechanism
Conformational characteristics and the adsorption behavior of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133°C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.
In situ structure and activity studies of an enzyme adsorbed on spectroscopically undetectable particles
Koutsopoulos, S. ; Tjeerdsma, A.M. ; Lieshout, J.F.T. van; Oost, J. van der; Norde, W. - \ 2005
Biomacromolecules 6 (2005)3. - ISSN 1525-7797 - p. 1176 - 1184.
archaeon pyrococcus-furiosus - solid-liquid interfaces - circular-dichroism - proteins - adsorption - endo-beta-1,3-glucanase - extremophiles - surfaces - features - sugar
The structural characteristics and the activity of a hyperthermophilic endoglucanase were investigated upon adsorption. Silica (hydrophilic) and Teflon (hydrophobic) surfaces were selected for the study. The materials were specially designed so that the interaction of the particles with light was negligible, and the enzyme conformation in the adsorbed state was monitored in situ. The adsorption isotherms were determined, and the adsorbed endoglucanase was studied using a number of spectroscopic techniques, enzymatic activity tests, and dynamic light scattering. Experiments were performed at pH values below, at, and above the isoelectric point of the enzyme. It was shown that the enzyme adsorbed on the hydrophobic surface of Teflon with higher affinity as compared to the hydrophilic silica nanoparticles. In all cases, adsorption was followed by (slight) changes in the secondary structure resulting in decreased -structural content. The changes were more profound upon adsorption on Teflon. The adsorbed enzyme remained active in the adsorbed state in spite of the structural changes induced when interacting with the surfaces
Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2005
Proteins : Structure, Function, and Bioinformatics 61 (2005)2. - ISSN 0887-3585 - p. 377 - 384.
archaeon pyrococcus-furiosus - crystal-structure - protein dynamics - 2.5-angstrom resolution - angstrom resolution - citrate synthase - thermostability - dehydrogenase - stability - fluctuations
The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25°C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein
Perspectives for the Industrial Enzymatic Production of Glycosides
Roode, B.M. de; Franssen, M.C.R. ; Padt, A. van der; Boom, R.M. - \ 2003
Biotechnology Progress 19 (2003)5. - ISSN 8756-7938 - p. 1391 - 1402.
immobilized beta-galactosidase - archaeon pyrococcus-furiosus - organic 2-phase system - packed-bed reactor - membrane reactor - alkyl glycosides - nonisothermal conditions - neisseria-polysaccharea - catalytic mechanism - glucoside synthesis
Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus, enzyme-catalyzed reactions are a good alternative. However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product. Unfortunately, a bioreactor for the commercial scale production of glycosides is not available. The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.
Enzyme inactivation due to Maillard reactions during oligosaccharide synthesis by a hyperthermophilic glycosidase : influence of enzyme immobilisation
Bruins, M.E. ; Thewessen, A.J.H. ; Janssen, A.E.M. ; Boom, R.M. - \ 2003
Journal of Molecular Catalysis. B, Enzymatic 21 (2003). - ISSN 1381-1177 - p. 31 - 34.
archaeon pyrococcus-furiosus - beta-glucosidase - purification
The extremely thermostable beta-glycosidase from Pyrococcus furiosus was used for the production of oligosaccharides with lactose as a substrate. Using a thermozyme made it possible to operate at higher reaction temperatures, and thus to increase the substrate concentration. This increased the substrate concentration and the subsequent lower water concentration suppressed hydrolysis and therefore improved the oligosaccharide yield. During the reaction, brown pigments were formed, caused by Maillard reactions. This changes the structure of the enzyme and causes faster inactivation of the enzyme, compared to normal inactivation by temperature. This faster inactivation is the main design criterion for the reaction system. Reduction of Maillard reactions can be done by altering the process conditions or through modification of the enzyme, either chemically or by altering the enzyme structure through genetic modifications. In this work, chemical modification of the enzyme was chosen by covalent immobilisation on Eupergit. Unfortunately, the immobilisation did not reduce Maillard reactivity. (C) 2002 Published by Elsevier Science B.V.
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