G Pettersson

Uppsala University, Uppsala, Uppsala, Sweden

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Publications (70)265.79 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The secondary structure pattern of a cellobiohydrolase from Trichoderma reesei was predicted using three different algorithms. The relative amounts of the different secondary structure classes derived by this procedure were in good agreement with the values determined by circular dichroism measurements. A twofold internal sequence homology with a repeat distance of approximately 200 residues is observed and possibly also a third, partial, repetition in the C-terminal region. The predicted secondary structure and hydrophobicity pattern show a similar repeat.
    European Journal of Allergy and Clinical Immunology 01/2009; 25(4):368 - 374. · 1.30 Impact Factor
  • Contrast Media & Molecular Imaging 01/2006; 1(2):90-90. · 2.87 Impact Factor
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    ABSTRACT: A recently developed method for quantitative assessment of regional lung ventilation was employed for the study of posture-dependent ventilation differences in rats. The measurement employed hyperpolarized (3)He MRI to detect the build-up of the signal intensity after increasing numbers of (3)He breaths, which allowed for computation of a regional ventilation parameter. A group of six anesthetized rats was studied in both supine and prone postures. Three-dimensional maps of the ventilation parameter were obtained with high spatial resolution (voxel volume approximately 2 mm(3)). Vertical (dorsal-ventral) gradients of the ventilation index, defined as the regional ventilation normalized by the average ventilation within the whole lung, were investigated. Variations in the regional distribution of the ventilation parameter, as well as of the ventilation index, could be detected, depending on the posture of the rats. In supine posture, ventilation was elevated in the dependent parts of the lungs, with a linear gradient of the ventilation index of -0.11 +/- 0.03 cm(-1). In prone posture, the distribution of ventilation was more uniform, with a significantly (P < 0.001) smaller gradient of the ventilation index of -0.01 +/- 0.02 cm(-1). It is concluded that the (3)He MRI-based method can detect and quantify regional ventilation gradients in animals as small as the rat and that these gradients depend on prone or supine posture of the animal.
    Journal of Applied Physiology 07/2005; 98(6):2259-67. · 3.48 Impact Factor
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    ABSTRACT: This paper reports the application of matrix-assisted UV-laser induced desorption (LID) mass spectrometry for the analysis of some extracellular multi-domain cellulolytic enzymes, in the molecular weight range 5000–90 000 u, produced by the fungi Phanerochaete chrysosporium and Trichoderma reesei. A high sensitivity (1–10pmol of protein), has been attained in obtaining the mass spectra of these highly glycosylated enzymes and some of their peptides, with carbohydrate content in the 10–40% range. The desorption of samples containing the enzymes mixed with a protein having an accurately known molecular weight—an internal calibrant—is also demonstrated. Spectra from complete enzymes and their functional domains are presented to explore the possibilities of employing LID for obtaining structural information.
    Rapid Communications in Mass Spectrometry 04/2005; 4(8):285 - 289. · 2.51 Impact Factor
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    ABSTRACT: The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.
    Biotechnology and Bioengineering 07/2004; 86(5):503-11. · 4.16 Impact Factor
  • Gunnar Johansson, Roland Isaksson, Göran Pettersson
    Methods in molecular biology (Clifton, N.J.) 02/2004; 243:307-15. · 1.29 Impact Factor
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    ABSTRACT: A fractal-like kinetics model was used to describe the synergistic hydrolysis of bacterial cellulose by Trichoderma reesei cellulases. The synergistic action of intact cellobiohydrolase Cel7A and endoglucanase Cel5A at low enzyme-to-substrate ratios showed an apparent substrate inhibition consistent with a case where two-dimensional (2-D) surface diffusion of the cellobiohydrolase is rate-limiting. The action of Cel7A core and Cel5A was instead consistent with a three-dimensional (3-D) diffusion-based mode of action. The synergistic action of intact Cel7A was far superior to that of the core at a high enzyme-to-substrate ratio, but this effect was gradually reduced at lower enzyme-to-substrate ratios. The apparent fractal kinetics exponent h obtained by nonlinear fit of hydrolysis data to the fractal-like kinetics analogue of a first-order reaction was a useful empirical parameter for assessing the rate retardation and its dependence on the reaction conditions.
    Biotechnology and Bioengineering 11/2003; 84(2):254-7. · 4.16 Impact Factor
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    ABSTRACT: A new strategy for a quantitative measurement of regional pulmonary ventilation using hyperpolarized helium-3 (3He) MRI has been developed. The method employs the build-up of the signal intensity after a variable number of (3)He breaths. A mathematical model of the signal dynamics is presented, from which the local ventilation, defined as the fraction of gas exchanged per breath within a given volume, is calculated. The model was used to create ventilation maps of coronal slices of guinea pig lungs. Ventilation values very close to 1 were found in the trachea and the major airways. In the lung parenchyma, regions adjacent to the hilum showed values of 0.6-0.8, whereas 0.2-0.4 was measured in peripheral regions. Monte Carlo simulations were used to investigate the accuracy of the method and its limitations. The simulations revealed that, at presently attainable signal-to-noise ratios, the ventilation parameter can be determined with a relative uncertainty of <5% over a wide range of values.
    Magnetic Resonance in Medicine 09/2002; 48(2):223-32. · 3.27 Impact Factor
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    Academic Radiology 09/2002; 9 Suppl 2:S507-10. · 1.91 Impact Factor
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    ABSTRACT: The fungus Phanerochaete chrysosporium was grown in a 10-l automatic fermenter using cellobiose as carbon source to monitor the induction of cellobiose dehydrogenase (CDH) and cellobiose quinone oxidoreductase (CBQ) enzymes, and to search for tentative cbq and cdh genes and their transcriptional products. After 24 h of induction, CDH was detected in the culture supernatant and a protein was recognized by a specific anti-CDH polyclonal antibody in the sonicated biomass. Northern blot experiments performed with several fungal RNA samples showed, after 24 h of induction, only one single species of an mRNA transcript corresponding in size to the cdh gene (2.5 kb) The relative amount of this transcript decreased as a function of time. Southern blot experiments done with genomic DNA and database search in the recently available genome information also ruled out the presence in this strain of a separate cbq gene distinct from the cdh gene. Taken together, these results demonstrated that CBQ originates from the cdh gene. Furthermore, it is not produced by differential splicing but by a posttranslational, predominantly intracellular, proteolytic cleavage.
    Biochimica et Biophysica Acta 07/2002; 1576(1-2):15-22. · 4.66 Impact Factor
  • Academic Radiology 06/2002; 9 Suppl 1:S62-4. · 1.91 Impact Factor
  • Journal of The American Chemical Society - J AM CHEM SOC. 04/2002; 112(11).
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    ABSTRACT: Cellobiose dehydrogenase (CDH) participates in the degradation of cellulose and lignin. The protein is an extracellular flavocytochrome with a b-type cytochrome domain (CYT(cdh)) connected to a flavodehydrogenase domain (DH(cdh)). DH(cdh) catalyses a two-electron oxidation at the anomeric C1 position of cellobiose to yield cellobiono-1,5-lactone, and the electrons are subsequently transferred from DH(cdh) to an acceptor, either directly or via CYT(cdh). Here, we describe the crystal structure of Phanerochaete chrysosporium DH(cdh) determined at 1.5 A resolution. DH(cdh) belongs to the GMC family of oxidoreductases, which includes glucose oxidase (GOX) and cholesterol oxidase (COX); however, the sequence identity with members of the family is low. The overall fold of DH(cdh) is p-hydroxybenzoate hydroxylase-like and is similar to, but also different from, that of GOX and COX. It is partitioned into an FAD-binding subdomain of alpha/beta type and a substrate-binding subdomain consisting of a seven-stranded beta sheet and six helices. Docking of CYT(cdh) and DH(cdh) suggests that CYT(cdh) covers the active-site entrance in DH(cdh), and that the resulting distance between the cofactors is within acceptable limits for inter-domain electron transfer. Based on docking of the substrate, cellobiose, in the active site of DH(cdh), we propose that the enzyme discriminates against glucose by favouring interaction with the non-reducing end of cellobiose.
    Journal of Molecular Biology 02/2002; 315(3):421-34. · 3.91 Impact Factor
  • Academic Radiology - ACAD RADIOL. 01/2002; 9(1).
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    ABSTRACT: Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, 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 I (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 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A 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.
    Journal of Molecular Biology 01/2002; 314(5):1097-111. · 3.91 Impact Factor
  • P Väljamäe, G Pettersson, G Johansson
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    ABSTRACT: A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.
    European Journal of Biochemistry 09/2001; 268(16):4520-6. · 3.58 Impact Factor
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    ABSTRACT: The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision.
    Analytical Biochemistry 04/2001; 290(2):245-50. · 2.58 Impact Factor
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    ABSTRACT: Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers. In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q. Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed. By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.
    Journal of Molecular Biology 02/2001; 305(1):79-93. · 3.91 Impact Factor
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    ABSTRACT: Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.
    Journal of Chromatography A 12/2000; 898(1):63-74. · 4.61 Impact Factor
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    ABSTRACT: The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide-dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non-phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non-phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non-phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse-phase high-pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV-visible spectroscopy.
    FEBS Letters 08/2000; 477(1-2):79-83. · 3.58 Impact Factor

Publication Stats

3k Citations
265.79 Total Impact Points

Institutions

  • 1984–2009
    • Uppsala University
      • • Department of Cell and Molecular Biology
      • • Department of Radiology, Oncology and Radiation Science
      • • Division of Analytical Pharmaceutical Chemistry
      Uppsala, Uppsala, Sweden
  • 2000–2005
    • Malmö University
      Malmö, Skåne, Sweden
    • Lund University
      Lund, Skåne, Sweden
  • 1996–2004
    • University of Tartu
      • Institute of Molecular and Cell Biology
      Tartu, Tartumaa, Estonia
    • Karl-Franzens-Universität Graz
      • Department of Chemistry
      Graz, Styria, Austria
  • 1995–2002
    • Center for Genetic Engineering and Biotechnology
      La Habana, Ciudad de La Habana, Cuba
    • VTT Technical Research Centre of Finland
      Esbo, Southern Finland Province, Finland
  • 1999–2000
    • KTH Royal Institute of Technology
      • Division of Wood Chemistry and Pulp Technology
      Stockholm, Stockholm, Sweden
  • 1993
    • Complutense University of Madrid
      Madrid, Madrid, Spain
  • 1988
    • Ghent University
      Gand, Flanders, Belgium