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Publications (6 of 6) Show all publications
Gustafsson, C., Vassiliev, S., Kürten, C., Syrén, P.-O. & Brinck, T. (2017). MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site. ACS Omega, 2(11), 8495-8506
Open this publication in new window or tab >>MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
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2017 (English)In: ACS Omega, ISSN 2470-1343, Vol. 2, no 11, p. 8495-8506Article in journal (Refereed) Published
Abstract [en]

Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
National Category
Biocatalysis and Enzyme Technology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-234939 (URN)10.1021/acsomega.7b01084 (DOI)000418744100113 ()
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180914

Available from: 2018-09-13 Created: 2018-09-13 Last updated: 2018-09-18Bibliographically approved
Eriksson, A., Kürten, C. & Syrén, P.-O. (2017). Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling. ChemBioChem (Print), 18(23), 2301-2305
Open this publication in new window or tab >>Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
2017 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 23, p. 2301-2305Article in journal (Refereed) Published
Abstract [en]

Terpenes represent one of the most diversified classes of natural products with potent biological activities. The key to the myriad of polycyclic terpene skeletons with crucial functions in organisms from all kingdoms of life are terpene cyclase enzymes. These biocatalysts enable stereospecific cyclization of relatively simple, linear, prefolded polyisoprenes by highly complex, partially concerted, electrophilic cyclization cascades that remain incompletely understood. Herein, additional mechanistic light is shed on terpene biosynthesis by kinetic studies in mixed H2O/D2O buffers of a classII bacterial ent-copalyl diphosphate synthase. Mass spectrometry determination of the extent of deuterium incorporation in the bicyclic product, reminiscent of initial carbocation formation by protonation, resulted in a large kinetic isotope effect of up to seven. Kinetic analysis at different temperatures confirmed that the isotope effect was independent of temperature, which is consistent with hydrogen tunneling.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2017
Keywords
biosynthesis, enzyme catalysis, isotope effects, kinetics, reaction mechanisms
National Category
Other Chemical Engineering
Identifiers
urn:nbn:se:kth:diva-220459 (URN)10.1002/cbic.201700443 (DOI)000417219500006 ()
Funder
Swedish Research Council, 621-2013-5138AFA Insurance, 17-359Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180104

Available from: 2018-01-04 Created: 2018-01-04 Last updated: 2018-09-13Bibliographically approved
Kürten, C., Carlberg, B. & Syren, P.-O. (2016). Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity. CATALYSTS, 6(6), Article ID 90.
Open this publication in new window or tab >>Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity
2016 (English)In: CATALYSTS, ISSN 2073-4344, Vol. 6, no 6, article id 90Article in journal (Refereed) Published
Abstract [en]

The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the alpha/beta phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

Place, publisher, year, edition, pages
MDPI AG, 2016
Keywords
enzyme promiscuity, enzyme catalysis, biocatalysis, reaction mechanisms, molecular modeling, amidase, esterase
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-189936 (URN)10.3390/catal6060090 (DOI)000378839100015 ()2-s2.0-84975302338 (Scopus ID)
Note

QC 20160728

Available from: 2016-07-28 Created: 2016-07-25 Last updated: 2018-09-18Bibliographically approved
Kürten, C. & Syren, P.-O. (2016). Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes. Journal of Visualized Experiments (107), Article ID e53168.
Open this publication in new window or tab >>Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
2016 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 107, article id e53168Article in journal (Refereed) Published
Abstract [en]

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Place, publisher, year, edition, pages
Journal of Visualized Experiments, 2016
Keywords
Chemistry, Issue 107, Enzyme catalysis, thermodynamics, water, dynamics, membrane protein, kinetics, transition state theory, protein engineering, hydrophobic effect
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-182168 (URN)10.3791/53168 (DOI)000368577400007 ()2-s2.0-84954533987 (Scopus ID)
Funder
Swedish Research Council, 621-2013-5138Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160218

Available from: 2016-02-18 Created: 2016-02-16 Last updated: 2018-09-18Bibliographically approved
Kürten, C., Uhlén, M. & Syrén, P.-O. (2015). Overexpression of functional human oxidosqualene cyclase in Escherichia coli. Protein Expression and Purification, 115, 46-53
Open this publication in new window or tab >>Overexpression of functional human oxidosqualene cyclase in Escherichia coli
2015 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 115, p. 46-53Article in journal (Refereed) Published
Abstract [en]

The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7 mg protein/g cells (hOSC in Pichia pastoris) after 48 h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9 mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
E. coli, Expression and purification, Membrane protein, Oxidosqualene cyclase, Triterpene cyclase
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-176183 (URN)10.1016/j.pep.2015.04.015 (DOI)000362623200007 ()2-s2.0-84941878054 (Scopus ID)
Funder
Swedish Research Council, 621-2013-5138
Note

QC 20151125

Available from: 2015-11-25 Created: 2015-11-02 Last updated: 2019-10-28Bibliographically approved
Kürten, C., Eriksson, A., Maddalo, G., Edfors, F., Uhlén, M. & Syrén, P.-O.Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme design.
Open this publication in new window or tab >>Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme design
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(English)Manuscript (preprint) (Other academic)
Keywords
Enzyme design, terpene cyclase, hydration, entropy
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-235186 (URN)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-09-17 Created: 2018-09-17 Last updated: 2018-09-18Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1685-4735

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