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  • 1.
    Engström, Joakim
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Stamm, Arne
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Tengdelius, Mattias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Fogelström, Linda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Cationic latexes of bio‐based hydrophobicmonomer Sobrerol methacrylate (SobMA)Manuscript (preprint) (Other academic)
  • 2.
    Engström, Karin
    et al.
    Stockholm Univ, Arrhenius Lab, Dept Organ Chem.
    Vallin, Michaela
    KTH, School of Biotechnology (BIO), Biochemistry.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bäckvall, Jan-E.
    Stockholm Univ, Arrhenius Lab, Dept Organ Chem.
    Mutated variant of Candida antarctica lipase B in (S)-selective dynamic kinetic resolution of secondary alcohols2011In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, no 1, p. 81-82Article in journal (Refereed)
    Abstract [en]

    An (S)-selective dynamic kinetic resolution of secondary alcohols, employing a mutated variant of Candida antarctica lipase B (CalB) gave products in 84-88% yield and in 90-97% ee.

  • 3.
    Eriksson, Adam
    et al.
    KTH, School of Chemical Science and Engineering (CHE).
    Kürten, Charlotte
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling2017In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 23, p. 2301-2305Article in journal (Refereed)
    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.

  • 4.
    Farhat, Wissam
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stamm, Arne
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Robert-Monpate, Maxime
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Ecole Super Chim Organ & Minerale, 1 Allee Reseau Jean Marie Buckmaster, F-60200 Compiegne, France.
    Biundo, Antonino
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Biocatalysis for terpene-based polymers2019In: Zeitschrift für Naturforschung C - A Journal of Biosciences, ISSN 0939-5075, E-ISSN 1865-7125, Vol. 74, no 3-4, p. 90-99Article in journal (Refereed)
    Abstract [en]

    Accelerated generation of bio-based materials is vital to replace current synthetic polymers obtained from petroleum with more sustainable options. However, many building blocks available from renewable resources mainly contain unreactive carbon-carbon bonds, which obstructs their efficient polymerization. Herein, we highlight the potential of applying biocatalysis to afford tailored functionalization of the inert carbocyclic core of multicyclic terpenes toward advanced materials. As a showcase, we unlock the inherent monomer reactivity of norcamphor, a bicyclic ketone used as a monoterpene model system in this study, to afford polyesters with unprecedented backbones. The efficiencies of the chemical and enzymatic Baeyer-Villiger transformation in generating key lactone intermediates are compared. The concepts discussed herein are widely applicable for the valorization of terpenes and other cyclic building blocks using chemoenzymatic strategies.

  • 5. Fink, Michael J.
    et al.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Redesign of water networks for efficient biocatalysis2017In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 37, p. 107-114Article, review/survey (Refereed)
    Abstract [en]

    Herein we highlight recent findings on the importance of water networks in proteins, and their redesign and reconfiguration as a new engineering strategy to generate enzymes with modulated binding affinity and improved catalytic versatility. Traditionally, enzyme engineering and drug design have focused on tailoring direct and favorable interactions between protein surfaces and ligands/transition states to achieve stronger binding, or an accelerated manufacturing of medicines, biofuels, fine chemicals and materials. In contrast, the opportunity to relocate water molecules in solvated binding pockets by protein design to improve overall energetics remains essentially unexplored, and fundamental understanding of the elusive processes involved is poor. Rewiring water networks in protein interiors impacts binding affinity, catalysis and the thermodynamic signature of biochemical processes through dynamic mechanisms, and thus has great potential to enhance binding specificity, accelerate catalysis and provide new reaction mechanisms and chemistry, that were not yet explored in nature.

  • 6.
    Fogelström, Linda
    et al.
    KTH, School of Engineering Sciences (SCI), Centres, VinnExcellence Center BiMaC Innovation. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Stamm, Arne
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Tengdelius, Mattias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH Royal Inst Technol, Fibre & Polymer Technol, Stockholm, Sweden..
    Malmström, Eva
    KTH Royal Inst Technol, Dept Fibre & Polymer Technol, Stockholm, Sweden..
    New chemo-enzymatic pathways for sustainable terpene-based polymeric materials2019In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 257Article in journal (Other academic)
  • 7.
    Gustafsson, Camilla
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Vassiliev, Serguei
    Department of Biological Sciences, Brock University, Ontario, Canada.
    Kürten, Charlotte
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Brinck, Tore
    MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site2017In: ACS Omega, ISSN 2470-1343, Vol. 2, no 11, p. 8495-8506Article in journal (Refereed)
    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.

  • 8. Hammer, Stephan C.
    et al.
    Dominicus, Joerg M.
    Syren, Per-Olof
    Universitaet Stuttgart, Germany.
    Nestl, Bettina M.
    Hauer, Bernhard
    Stereoselective Friedel-Crafts alkylation catalyzed by squalene hopene cyclases2012In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 68, no Copyright (C) 2013 American Chemical Society (ACS). All Rights Reserved., p. 7624-7629Article in journal (Refereed)
    Abstract [en]

    In org. synthesis the Friedel-Crafts alkylation is of eminent importance, as it is a key reaction in many synthetic routes. A general access to enzymic Friedel-Crafts alkylations would be very beneficial due to the high selectivity of biocatalysts. We used designed polyprenyl Ph ethers to specifically address this reaction by using squalene hopene cyclases as catalysts. Polycyclic products with arom. rings constituting important biol. active compds. were obtained. Our results demonstrate that squalene hopene cyclases can be utilized for Friedel-Crafts alkylations and reveal the potential of these enzymes for chiral Bronsted acid catalysis.

  • 9. Hammer, Stephan C.
    et al.
    Syren, Per-Olof
    Universitaet Stuttgart, Germany.
    Seitz, Miriam
    Nestl, Bettina M.
    Hauer, Bernhard
    Squalene hopene cyclases: highly promiscuous and evolvable catalysts for stereoselective CC and CX bond formation2013In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 17, no 2, p. 293-300Article in journal (Refereed)
    Abstract [en]

    A review. We review here how the inherent promiscuous nature, as well as the evolvability of terpene cyclase enzymes enables new applications in chem. We mainly focus on squalene hopene cyclases, class II triterpene synthases that use a proton-initiated cationic polycyclization cascade to form carbopolycyclic products. We highlight recent findings to demonstrate that these enzymes are capable of activating different functionalities other than the traditional terminal isoprene CC-group as well as being compatible with a wide range of nucleophiles beyond the 'ene-functionality'. Thus, squalene hopene cyclases demonstrate a great potential to be used as a toolbox for general Bronsted acid catalysis.

  • 10. Hammer, Stephan C.
    et al.
    Syrén, Per-Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Hauer, Bernhard
    Substrate Pre-Folding and Water Molecule Organization Matters for Terpene Cyclase Catalyzed Conversion of Unnatural Substrates2016In: CHEMISTRYSELECT, ISSN 2365-6549, Vol. 1, no 13, p. 3589-3593Article in journal (Refereed)
    Abstract [en]

    Terpene cyclase enzymes have recently been challenged with terpene substrate derivatives to generate additional chemical complexity beyond to what is currently found in nature. Herein, molecular dynamics and biocatalysis are used to shed light on the flexibility and inherent limitation of a triterpene cyclase in converting unnatural substrates. Our studies suggest that populating binding modes which allows for concerted reaction pathways is a key element towards an expanded substrate scope and new chemistries displayed by terpene cyclases. Additionally, we show that the spatial organization of water, which is influenced by both the substrate architecture as well as the active site geometry, controls the product selectivity. This highlights that activity and selectivity displayed by terpene cyclases acting on unnatural substrates is particularly difficult to predict, since they depend on various parameters.

  • 11. Hammer, Stephan
    et al.
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
    Hauer, Bernhard
    Substrate Pre-Folding and Water Molecule Organization Matters for Terpene Cyclase Catalyzed Conversion of Unnatural Substrates2016In: ChemistrySelect, ISSN 2365-6549, Vol. 1, p. 3589-3593Article in journal (Refereed)
    Abstract [en]

    Terpene cyclase enzymes have recently been challenged with terpene substrate derivatives to generate additional chemical complexity beyond to what is currently found in nature. Herein, molecular dynamics and biocatalysis are used to shed light on the flexibility and inherent limitation of a triterpene cyclase in converting unnatural substrates. Our studies suggest that populating binding modes which allows for concerted reaction pathways is a key element towards an expanded substrate scope and new chemistries displayed by terpene cyclases. Additionally, we show that the spatial organization of water, which is influenced by both the substrate architecture as well as the active site geometry, controls the product selectivity. This highlights that activity and selectivity displayed by terpene cyclases acting on unnatural substrates is particularly difficult to predict, since they depend on various parameters. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  • 12.
    Hedfors, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Takwa, Mohamad
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Selectivity towards itaconic acid esters by Candida antarctica lipase B and variantsManuscript (preprint) (Other academic)
  • 13.
    Hendil-Forssell, Peter
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Syren, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Exploring water as building bricks in enzyme engineering2015In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 51, no 97, p. 17221-17224Article in journal (Refereed)
    Abstract [en]

    A novel enzyme engineering strategy for accelerated catalysis based on redesigning a water network through protein backbone deshielding is presented. Fundamental insight into the energetic consequences associated with the design is discussed in the light of experimental results and computer simulations. Using water as biobricks provides unique opportunities when transition state stabilisation is not easily attained by traditional enzyme engineering.

  • 14.
    Hendrikse, N. M.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Stromberg, P.
    Nordling, E.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Redesign of biosynthetic enzymes using ancestral sequence reconstruction2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, p. 86-87Article in journal (Refereed)
  • 15.
    Hendrikse, Natalie
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.
    Charpentier, Gwenaelle
    KTH, Centres, Science for Life Laboratory, SciLifeLab. ESCOM, 1 Allee Reseau Jean Marie Buckmaster, F-60200 Compiegne, France..
    Nordling, Erik
    Swedish Orphan Biovitrum AB, Stockholm, Sweden..
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.
    Ancestral diterpene cyclases show increased thermostability and substrate acceptance2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 24, p. 4660-4673Article in journal (Refereed)
    Abstract [en]

    Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 degrees C. Ancestral spiroviolene synthases catalyze cyclization of the natural C-20-substrate geranylgeranyl diphosphate (GGPP) and also accept C-15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., k(cat)/K-M for GGPP over k(cat)/K-M for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.

  • 16.
    Kürten, Charlotte
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Carlberg, Bengt
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Syren, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity2016In: CATALYSTS, ISSN 2073-4344, Vol. 6, no 6, article id 90Article in journal (Refereed)
    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.

  • 17.
    Kürten, Charlotte
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Eriksson, Adam
    Maddalo, Gianluca
    Edfors, Fredrik
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme designManuscript (preprint) (Other academic)
  • 18.
    Kürten, Charlotte
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Syren, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes2016In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 107, article id e53168Article in journal (Refereed)
    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.

  • 19.
    Kürten, Charlotte
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Overexpression of functional human oxidosqualene cyclase in Escherichia coli2015In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 115, p. 46-53Article in journal (Refereed)
    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.

  • 20.
    Malmström, Eva
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Fogelström, Linda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Stamm, Arne
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Tengdelius, Mattias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Biundo, Antonino
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Sustainable terpene-based polymeric materials2019In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 257Article in journal (Other academic)
  • 21. Marton, Z.
    et al.
    Leonard-Nevers, V.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry.
    Bauer, C.
    Lamare, S.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Tranc, V.
    Graber, M.
    Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity2010In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 65, no 1-4, p. 11-17Article in journal (Refereed)
    Abstract [en]

    Two different parts of Candida antarctica lipase B (stereospecificity pocket at the bottom of the active site and hydrophobic tunnel leading to the active site) were redesigned by single- or double-point mutations, in order to better control and improve enzyme enantioselectivity toward secondary alcohols. Single-point isosteric mutations of Ser47 and Thr42 situated in the stereospecificity pocket gave rise to variants with doubled enantioselectivity toward pentan-2-ol, in solid/gas reactor. Besides, the width and shape of the hydrophobic tunnel leading to the active site was modified by producing the following single-point mutants: Ile189Ala, Leu278Val and Ala282Leu. For each of these variants a significant modification of enantioselectivity was observed compared to wild-type enzyme, indicating that discrimination of the enantiomers by the enzyme could also arise from their different accessibilities from the enzyme surface to the catalytic site. (C) 2010 Elsevier B.V. All rights reserved.

  • 22. Pavlidis, I. V.
    et al.
    Hendrikse, Natalie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chapter 5: Computational Techniques for Efficient Biocatalysis2018In: RSC Catalysis Series, no 32, p. 119-152Article in journal (Refereed)
    Abstract [en]

    Addressing some of the most challenging problems that we face today, including depletion of natural resources, sustainable energy production and the generation of green polymeric materials by the biocatalytic upcycling of renewable synthons, requires an expansion of the current available biochemical reaction space. Creating biocatalysts harboring novel chemistries - whether inside or outside the cell - is dependent on the discovery of novel enzymes and metabolic pathways, together with the de novo design of enzymes and directed evolution. Herein we review the high potential of using bioinformatics and in silico computer modelling tools to guide protein engineering and to enhance our fundamental understanding of biocatalysis. Following an overview of technical considerations and the current state-of-the art in sequence- and structure-based protein engineering methodologies, we highlight recent successful examples of their implementation in biocatalysis and synthetic biology. Moreover, we discuss how selected computational tools in concert with experimental biocatalysis could decipher how the sequence, structure and dynamics of proteins dictate their function. Using the methodologies discussed in this chapter, an accelerated biocatalytic manufacturing of chemicals, pharmaceuticals, biofuels and monomeric building blocks is envisioned.

  • 23. Seitz, Miriam
    et al.
    Syrén, Per-Olof
    Steiner, Lisa
    Klebensberger, Janosch
    Nestl, Bettina
    Hauer, Bernhard
    Synthesis of heterocyclic terpenoids by promiscuous squalene-hopene cyclases2013In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 14, p. 436-439Article in journal (Refereed)
  • 24.
    Stamm, Arne
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Biundo, Antonino
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schmidt, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brücher, Jörg
    Holmen AB, Dev, S-89180 Östersund, Sweden.
    Lundmark, Stefan
    Perstorp AB, Innovat, Perstorp Ind Pk, S-28480 Perstorp, Sweden.
    Olsén, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Fogelström, Linda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center.
    Bornscheuer, Uwe T
    Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Dept Biotechnol & Enzyme Catalysis, Felix Hausdorff Str 4, D-17487 Greifswald, Germany.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    A retrobiosynthesis-based route to generate pinene-derived polyesters2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, p. 1664-1671Article in journal (Refereed)
    Abstract [en]

    Significantly increased production of biobased polymers is aprerequisite to replace petroleum-based materials towardsreaching a circular bioeconomy. However, many renewablebuilding blocks from wood and other plant material are notdirectly amenable for polymerization, due to their inert backbonesand/or lack of functional group compatibility with thedesired polymerization type. Based on a retro-biosyntheticanalysis of polyesters, a chemoenzymatic route from (@)-apinenetowards a verbanone-based lactone, which is furtherused in ring-opening polymerization, is presented. Generatedpinene-derived polyesters showed elevated degradation andglass transition temperatures, compared with poly(e-decalactone),which lacks a ring structure in its backbone. Semirationalenzyme engineering of the cyclohexanone monooxygenasefrom Acinetobacter calcoaceticus enabled the biosynthesis ofthe key lactone intermediate for the targeted polyester. As aproof of principle, one enzyme variant identified from screeningin a microtiter plate was used in biocatalytic upscaling,which afforded the bicyclic lactone in 39% conversion in shakeflask scale reactions.

  • 25.
    Stamm, Arne
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Tengdelius, Mattias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Schmidt, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Engström, Joakim
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Centres, Wallenberg Wood Science Center. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Fogelström, Linda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Malmström, Eva
    KTH, School of Engineering Sciences (SCI), Centres, VinnExcellence Center BiMaC Innovation.
    Chemo- enzymatic pathways toward pinene- based renewable materials2019In: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 21, no 10, p. 2720-2731Article in journal (Refereed)
    Abstract [en]

    Sobrerol methacrylate (SobMA) was synthesized and subsequently polymerized using different chemical and enzymatic routes. Sobrerol was enzymatically converted from -pinene in a small model scale by a Cytochrome P450 mutant from Bacillus megaterium. Conversion of sobrerol into SobMA was performed using both classical ester synthesis, i.e., acid chloride-reactions in organic solvents, and a more green approach, the benign lipase catalysis. Sobrerol was successfully esterified, leaving the tertiary alcohol and ene to be used for further chemistry. SobMA was polymerized into PSobMA using different radical polymerization techniques, including free radical (FR), controlled procedures (Reversible Addition Fragmentation chain-Transfer polymerization, (RAFT) and Atom Transfer Radical Polymerization (ATRP)) as well as by enzyme catalysis (horseradish peroxidase-mediated free radical polymerization). The resulting polymers showed high glass-transition temperatures (T-g) around 150 degrees C, and a thermal degradation onset above 200 degrees C. It was demonstrated that the T-g could be tailored by copolymerizing SobMa with appropriate methacrylate monomers and that the Flory-Fox equation could be used to predict the T-g. The versatility of PSobMA was further demonstrated by forming crosslinked thin films, either using the ene'-functionality for photochemically initiated thiol-ene'-chemistry, or reacting the tertiary hydroxyl-group with hexamethoxymethylmelamine, as readily used for thermally curing coatings systems.

  • 26. Syren, Per-Olof
    The solution of nitrogen inversion in amidases2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 13, p. 3069-3083Article in journal (Refereed)
    Abstract [en]

    An important mechanistic aspect of enzyme-catalyzed amide bond hydrolysis is the specific orientation of the lone pair of the N atom of the scissile amide bond during catalysis. As discussed in the literature during the last decades, stereoelectronic effects cause the single lone pair in the formed tetrahedral intermediate to be situated in a non-productive conformation in the enzyme active site and hence N atom inversion or rotation is necessary. By discussing recent mechanistic findings in the literature relevant for the conformation of the lone pair of the reacting amide N atom, it is demonstrated that Nature has evolved at least 2 catalytic strategies to cope with the stereoelectronic constraints inherent to amide bond hydrolysis regardless of the fold or catalytic mechanism. One soln. to the inversion problem is to stabilize the transition state of inversion by H-bond formation; another is to introduce a concerted proton shuttle mechanism that avoids inversion and delivers a hydrogen to the lone pair. Here, by using mol. modeling it was demonstrated that the H-bond strategy is general and can be expanded to include many amidases/proteases with important metabolic functions, including the proteasome. Some examples of the proton shuttle mechanism are also mentioned. To complete the picture of efficient enzyme-catalyzed amide bond hydrolysis, general interactions in the active site of these catalysts were discussed. An expanded knowledge of the prerequisites of efficient amide bond hydrolysis beyond the oxyanion hole and the catalytic dyad/triad will be of importance for enzyme and drug design. [on SciFinder(R)]

  • 27.
    Syren, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Lindgren, Ebba
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hoeffken, Hans Wolfgang
    Branneby, Cecilia
    Maurer, Steffen
    Hauer, Bernhard
    Hult, Karl
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Biotechnology (BIO), Biochemistry.
    Increased activity of enzymatic transacylation of acrylates through rational design of lipases2010In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 65, no 1-4, p. 3-10Article in journal (Refereed)
    Abstract [en]

    A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k(cat) three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H2SO4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the pi-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate. (C) 2009 Elsevier B.V. All rights reserved.

  • 28. Syren, Per-Olof
    et al.
    Rozkov, Aleksei
    Schmidt, Stefan R.
    Stroemberg, Patrik
    Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump2007In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 856, p. 68-74Article in journal (Refereed)
    Abstract [en]

    A parallel chromatog. procedure for the purifn. of milligram amts. of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quaternary ammonium group with respect to elution and regeneration properties and the 75 cm2 Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimized. By introducing this step into the protocol, endotoxin levels could be reduced approx. 100-fold to ≤5 EU/mg plasmid. The parallel procedure was set up on a multi-channel peristaltic pump and evaluated with four different vectors (2.7-11.5 kbp). Starting with 5-10 g of E. coli cell paste (wet wt.) generally satd. the membrane adsorber, resulting in plasmid DNA yields close to 10 mg. [on SciFinder(R)]

  • 29.
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Enzymatic Hydrolysis of Tertiary Amide Bonds by anti Nucleophilic Attack and Protonation2018In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 83, no 21, p. 13543-13548Article in journal (Refereed)
    Abstract [en]

    The molecular mechanisms conferring high resistance of planar tertiary amide bonds to hydrolysis by most enzymes have remained elusive. To provide a chemical explanation to this unresolved puzzle, UB3LYP calculations were performed on an active site model of Xaa-Pro peptidases. The calculated reaction mechanism demonstrates that biocatalysts capable of tertiary amide bond hydrolysis capitalize on anti nucleophilic attack and protonation of the amide nitrogen, in contrast to the traditional syn displayed by amidases and proteases acting on secondary amide bonds.

  • 30.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry.
    On electrostatic effects, minimal motion and other catalytic strategies used by enzymes2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes are powerful biocatalysts that provide rate accelerations of up to 1019 fold compared to the corresponding uncatalyzed reaction in solution. The origin of the remarkable performance displayed by enzymes has fascinated and puzzled researchers for over a hundred years. It is clear that the catalytic effect is a consequence of the higher degree of transition state stabilization for the enzyme catalyzed reaction compared to the corresponding uncatalyzed reaction. It is still not well understood exactly how this transition state stabilization occurs and the relative importance of various catalytic effects are discussed. Catalytic effects involving electrostatics, near attack conformers, dynamic effects and an economy in atomic motion are discussed in this thesis.

    The importance of electrostatic effects is corroborated in this thesis. A single hydrogen bond in transition state constitutes an important difference between amidases and esterases. A hydrogen bond in transition state is found in all sixteen analyzed amidases representing ten different reaction mechanisms and eleven different folding families. The hydrogen bond is shown to be either substrate assisted or enzyme assisted. The role of this hydrogen bond is to assist nitrogen inversion in amidases. Esterases lack this interaction in transition state and therefore they are very poor catalysts in the hydrolysis of amides. Electrostatic interactions are found to facilitate proton transfer that enhances the rate of lipase catalyzed N-acylation of amino alcohols.

    In this thesis electrostatic effects in the substrate are shown to be important for the lipase catalyzed transacylation of acrylates The α,β-double bond present in acrylates introduce electronic effects that has the consequence of restricting the conformational freedom of the substrate in its ground state to two flat conformations, s-cis and s-trans. It is shown that acrylates form near attack conformers (NACs) from their ground state s-cis/s-trans planar conformations. The ability of the enzyme to accommodate such apparent s-cis/s-trans substrate conformations dictates the probability to form productive transition states and thus the reaction rate.

    Dynamic effects are important in enzymes. In this thesis it is found that a point mutation increases the flexibility of a neighbouring residue in Candida antarctica lipase B. This allows the mutated enzyme to explore conformations not accessible for the wild-type enzyme. The dynamics has the effect to decrease steric interactions in transition state with concomitant rate increase for the transacylation of methyl methacrylate.

    In this thesis an economy of atomic motion during enzyme catalysis is observed. Nitrogen inversion in amidases constitutes an interesting example. A rotation as part of the reaction mechanism for amide bond hydrolysis would involve much more motion.

  • 31.
    Syrén, Per-Olof
    University of Stuttgart.
    The solution to nitrogen inversion in amidases2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, p. 3069-3083Article in journal (Refereed)
    Abstract [en]

    An important mechanistic aspect of enzyme catalyzed amide bond hydroly-sis is the specific orientation of the lone pair of the nitrogen of the scissileamide bond during catalysis. As discussed in the literature during the lastdecades, stereoelectronic effects cause the single lone pair in the formedtetrahedral intermediate to be situated in a non-productive conformationin the enzyme active site and hence nitrogen inversion or rotation is neces-sary. By discussing recent mechanistic findings in the literature relevant forthe conformation of the lone pair of the reacting amide nitrogen atom, itwill be demonstrated that nature has evolved at least two catalytic strate-gies to cope with the stereoelectronic constraints inherent to amide bondhydrolysis regardless of the fold or catalytic mechanism. One solution tothe inversion problem is to stabilize the transition state of inversion byhydrogen bond formation; another is to introduce a concerted proton shut-tle mechanism that avoids inversion and delivers a hydrogen to the lonepair. By using molecular modeling it is demonstrated that the H-bondstrategy is general and can be expanded to include many amidases/prote-ases with important metabolic functions, including the proteasome. Someexamples of the proton shuttle mechanism will also be mentioned. To com-plete the picture of efficient enzyme catalyzed amide bond hydrolysis, gen-eral interactions in the active site of these catalysts will be discussed. Anexpanded knowledge of the prerequisites of efficient amide bond hydrolysisbeyond the oxyanion hole and the catalytic dyad/triad will be of impor-tance for enzyme and drug design.

  • 32.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Understanding esterase and amidase reaction specificities by molecular modeling2016In: Understanding Enzymes: Function, Design, Engineering and Analysis, Pan Stanford Publishing, 2016, p. 523-558Chapter in book (Other academic)
  • 33.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Understanding esterase and amidase reaction specificities by molecular modelling2016In: Understanding enzymes; Function, Design, Engineering and Analysis, Pan Standford Publishing , 2016, p. 523-Chapter in book (Refereed)
  • 34.
    Syrén, Per-Olof
    et al.
    Institute of Technical Biochemistry, University of Stuttgart, Germany.
    Hammer, Stephan C.
    Claasen, Birgit
    Hauer, Bernhard
    Entropy is Key to the Formation of Pentacyclic Terpenoids by Enzyme-Catalyzed Polycyclization2014In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 53, no 19, p. 4845-4849Article in journal (Refereed)
    Abstract [en]

    Polycyclizations constitute a cornerstone of chemistry and biology. Multicyclic scaffolds are generated by terpene cyclase enzymes in nature through a carbocationic polycyclization cascade of a prefolded polyisoprene backbone, for which electrostatic stabilization of transient carbocationic species is believed to drive catalysis. Computational studies and site-directed mutagenesis were used to assess the contribution of entropy to the polycyclization cascade catalyzed by the triterpene cyclase from A. acidocaldarius. Our results show that entropy contributes significantly to the rate enhancement through the release of water molecules through specific channels. A single rational point mutation that results in the disruption of one of these water channels decreased the entropic contribution to catalysis by 60kcalmol(-1). This work demonstrates that entropy is the key to enzyme-catalyzed polycyclizations, which are highly relevant in biology since 90% of all natural products contain a cyclic subunit.

  • 35.
    Syrén, Per-Olof
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Henche, S.
    Eichler, A.
    Nestl, B. M.
    Hauer, B.
    Squalene-hopene cyclases: evolution, dynamics and catalytic scope2016In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 41, p. 73-82Article in journal (Refereed)
    Abstract [en]

    Herein we highlight recent mechanistic findings on the impact of solvent dynamics on catalysis displayed by squalene-hopene cyclases (SHCs). These fascinating biocatalysts that appeared early during the evolution of terpene biosynthetic machineries exploit a catalytic aspartic acid donating the anti-oriented proton to the terminal C. C double bond of pre-folded isoprenoid substrates. We review how the unusual strength of this Brønsted acid can be used to harness a plethora of non-natural protonation-driven reactions in a plastic enzyme fold. Moreover, recent results underline how the reaction termination by deprotonation or water addition is governed by the spatial location of water in the active site. Site-directed mutagenesis of amino acids located in the hydrophobic binding pocket allows for the generation of novel catalytic function by active site reshaping with relatively small enzyme libraries. A deepened understanding of triterpene cyclase dynamics in concert with chemical expertise thus have a great potential to allow for the biocatalytic manufacturing of tailored building bricks that would expand the chemical repertoire currently found in nature.

  • 36.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hendil-Forssell, Peter
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Aumailley, Lucie
    Besenmatter, Werner
    Gounine, Farida
    Svendsen, Allan
    Martinelle, Mats
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 5, p. 645-648Article in journal (Refereed)
  • 37.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Amidases have a hydrogen bond that facilitates nitrogen inversion but esterases have not2011In: ChemCatChem, ISSN 1867-3899, Vol. 3, no 5, p. 853-860Article in journal (Refereed)
    Abstract [en]

    The fact that proteases/amidases can hydrolyze amides efficiently whereas esterases can not has been discussed during the last decades. By using molecular modeling we have found a hydrogen bond in the transition state for protease/amidase catalyzed hydrolysis of peptides and amides donated by the scissile NH-group of the substrate. The hydrogen-bond acceptor was found either in the enzyme (enzyme assisted) or in the substrate (substrate assisted). This new interaction with the NH-hydrogen in the transition state (TS) was found in sixteen proteases/amidases, which represent ten different reaction mechanisms and eleven different folding families. Esterases lack this interaction and, therefore, they are slow in hydrolyzing amides. By mimicking the substrate-assisted catalysis found in amidases we were able to shift reaction specificity of amide over ester synthesis of Candida antarctica lipase B one hundred fold. We propose that the hydrogen bond facilitates nitrogen inversion in amidases.

  • 38.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Substrate Conformations Set the Rate of Enzymatic Acrylation by Lipases2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 6, p. 802-810Article in journal (Refereed)
    Abstract [en]

    Acrylates represent a class of 4-unsaturated compounds of high industrial importance. We investigated the influence of substrate conformations on the experimentally determined reaction rates of the enzyme-catalysed transacylation of methyl acrylate and derivatives by ab initio DFT B3LYP calculations and molecular dynamics simulations. The results supported a least-motion mechanism upon the sp(2) to sp(3) substrate transition to reach the transition state in the enzyme active site. This was in accordance with our hypothesis that acrylates form productive transition states from their low-energy s-sis/s-trans conformations. Apparent k(cat) values were measured for Candida antarctica lipase B (CALB), Humicola insolens cutinase and Rhizomucor miehei lipase and were compared to results from computer simulations. More potent enzymes for acryltransfer, such as the CALB mutant V190A and acrylates with higher turnover numbers, showed elevated populations of productive transition states.

  • 39.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Le Joubioux, Florian
    Ben Henda, Yesmine
    Maugard, Thierry
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Graber, Marianne
    Proton Shuttle Mechanism in the Transition State of Lipase-Catalyzed N-Acylation of Amino Alcohols2013In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 5, no 7, p. 1842-1853Article in journal (Refereed)
    Abstract [en]

    An increased reaction rate for lipase-catalyzed N-acylation of amino alcohols relative to that of monofunctionalized amines can be explained by a hydrogen shuttling mechanism that avoids nitrogen inversion in the transition state. The mechanism does not involve acyl migration from an ester intermediate that would be formed first, an explanation that permeates the literature. Our suggested reaction mechanism is dependent on the preference of amino alcohols to form intramolecular hydrogen bonds and the capability of the enzyme to accommodate and exploit the specific hydrogen bonding pattern provided by the ligand during catalysis. Our proposed proton shuttle mechanism involves the transfer of two protons in the transition state concomitant with a nucleophilic attack on the acyl enzyme and provides an explanation for the high reaction rate and chemoselectivity for lipase-catalyzed N-acylation of amino alcohols. Moreover, the proton shuttle mechanism explains the increased reaction rate for the enzyme-catalyzed N-acylation of diamines and of methoxy-2-propylamine, for which O- to N-acyl migration is impossible. A linear free-energy relationship analysis based on the experimental results showed that all of our investigated difunctionalized amine substrates afforded a substrate-assisted rate acceleration of the N-acylation by the same reaction mechanism. Furthermore, the results of the analysis were consistent with partial proton transfer in the rate-limiting transition state, which further supports our suggested proton shuttle mechanism.

  • 40.
    Syrén, Per-Olof
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Le Joubioux, Florian
    Henda, Jesmine Ben
    Maugard, Thierry
    Graber, Marianne
    Proton transfer in amino alcohols in transition state of lipase catalyzed N-acylation.Manuscript (preprint) (Other academic)
  • 41.
    Vallin, Michaela
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Biochemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Mutant Lipase-Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec-Alcohols: A Thermodynamic Analysis of Enantioselectivity2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 3, p. 411-416Article in journal (Refereed)
    Abstract [en]

    The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

1 - 41 of 41
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