Lpmos) ✉ Riin Kont1, Bastien Bissaro 2,3, Vincent G

Total Page:16

File Type:pdf, Size:1020Kb

Lpmos) ✉ Riin Kont1, Bastien Bissaro 2,3, Vincent G ARTICLE https://doi.org/10.1038/s41467-020-19561-8 OPEN Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs) ✉ Riin Kont1, Bastien Bissaro 2,3, Vincent G. H. Eijsink 2 & Priit Väljamäe 1 Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of 1234567890():,; LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic prop- erties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, 5 6 −1 −1 with kcat/KmH2O2 values in the order of 10 –10 M s . Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases. 1 Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. 2 Faculty of Chemistry, Biotechnology and Food Science, NMBU—Norwegian University of Life Sciences, Ås, Norway. 3 INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques, 13009 Marseille, France. ✉ email: [email protected] NATURE COMMUNICATIONS | (2020) 11:5786 | https://doi.org/10.1038/s41467-020-19561-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19561-8 ytic polysaccharide monooxygenases (LPMOs) are mono- is a non-heme-iron peroxidase rather than an oxidase, we have Lcopper enzymes involved in degradation of polysaccharides, developed a theoretical framework and an experimental setup including cellulose and chitin, which are the most abundant to kinetically characterize a so far non-characterized and very polysaccharides in Nature. Their oxidative mechanism was dis- important LPMO reaction, the cellulolytic peroxygenase reac- covered in 2010 by showing that the chitin-binding protein tion. We have addressed this issue by setting up competition CBP211,2 of the bacterium Serratia marcescens (here referred to as experiments with a kinetically well characterized reference SmAA10A) catalyzes oxidative cleavage of β-1,4 glycosidic bonds enzyme and an LPMO of interest, to derive k /K values cat mH2O2 in chitin, while generating C1-oxidized oligosaccharide products3. for LPMOs acting on soluble cellooligosaccharides as well as The reaction was shown to be dependent on the presence of O2 insoluble cellulose (Avicel). One of the two reference enzymes and reductant3. Ten years of intensive research has revealed the in this study was a chitin-active bacterial LPMO, SmAA10A, for – presence of LPMOs in most kingdoms of life3 7 and today, these which detailed kinetic data exist thanks to the availability of enzymes are classified within seven families of auxiliary activities unique, but not generally available, experimental tools that (AA)8 in the database of carbohydrate-active enzymes9, with include the use of 14C-labeled chitin nanowhiskers (CNWs)14. families AA9, from fungal origin, and AA10, primarily from The other reference enzyme was readily available horseradish bacterial origin, being the best studied. To date, many LPMOs, peroxidase (HRP) acting on two different, both readily avail- acting on various oligo- and polysaccharides, and using different able, substrates, N-acetyl-3,7-dihydroxyphenoxazine (Amplex regioselectivities of oxidation, have been identified10. Red, AR) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic While structurally and functionally well characterized7,11, kinetic acid) diammonium salt (ABTS). The cellulose-active LPMOs studies of LPMOs are scarce. The scarcity of kinetic data likely studied were fungal family AA9 LPMOs from Trichoderma reflects the numerous experimental challenges associated with reesei (TrAA9A) and Neurospora crassa (NcAA9C), as well as a quantitative characterization of LPMO functionality12.Thecom- bacterial AA10 LPMO from Streptomyces coelicolor (ScAA10C, plexity is exemplified by the recent discovery that LPMOs may use formerly also known as CelS2). 13–20 H2O2, rather than, or even instead of, O2, as co-substrate . Regardless of the mechanism, LPMOs need an external electron donor for catalysis. In the H2O2-driven reaction, the reductant is Results used only in a “priming-reduction” of the Cu(II) resting state to the Two enzymes competing for the same co-substrate—kinetic 13,18,21 catalytically active Cu(I) form whereas in the O2-driven foundation. Let us consider a system where two enzymes, the reaction the reductant is consumed in stoichiometric amounts (i.e., reference enzyme and the enzyme of interest compete for the 10,22 delivery of two electrons per glycosidic bond cleavage) .The H2O2 co-substrate (Fig. 1). The substrate of the reference enzyme auto oxidation of reductants by O2 often leads to the formation of is designated as Sref and that of the enzyme of interest as S. The H2O2 and this complicates the interpretation of kinetic data. Fur- experimental approach used in this study requires that the values thermore, LPMOs that are free from substrate have both reductant of the kinetic parameters of the reference enzyme are known. To oxidase23 and peroxidase16,18 activity where the oxidase activity this end, we used the well-characterized chitin-active SmAA10A 17,23–25 leads to the formation of H2O2 . as well as HRP as reference enzymes (see below). To date only two detailed kinetic studies of LPMO action on Enzyme reactions involving two substrates may follow a carbohydrate substrates are available. Kuusk et al.14 have shown ternary complex (both substrates are bound to the enzyme) or a that the H2O2-driven oxidation of insoluble chitin by (bacterial) ping-pong mechanism. Depending on the order of the binding of 6 −1 −1 SmAA10A has a kcat/KmH O in the order of 10 M s ,whereas substrates the mechanisms involving a ternary complex are 26 2 2 Hangasky et al. have shown that O2-driven oxidation of soluble further grouped as random order or ordered mechanisms. In the cellohexaosebyanLPMOfromthefungusMyceliophthora ther- absence of products, the steady-state rates of H2O2 consumption mophilia (MtPMO9E) has a k /K in the order of 103 M−1 s−1. cat mO2 (d[H2O2]/dt) for an enzyme obeying an ordered (with S being the While the relative importance of the O2-andH2O2-driven reaction first substrate to bind) ternary complex or a ping-pong mechanisms remains a subject of debate15, available data clearly show that the peroxygenase reaction is much faster14,18,22,27. Unfortunately, due to a lack of suitable methods, so far, there are no ref ref Measurement S S -O Interest solid kinetic data for one of the most important LPMO reactions, V = f ([LP MO ]) namely oxidative degradation of insoluble cellulose. ref 27 28–30 In Nature as well as in industrial applications , LPMOs Eref involved in lignocellulose degradation operate in redox-active (Sm AA10A; HRP) ref environments where both potential co-substrates, O2 and H2O2,as kcat H2O2 Competition H2O ref well as multiple compounds and/or enzymes interacting with Km these co-substrates, are present. For example, the secretomes of Interest fungi growing on lignocellulosic biomass contain, next to LPMOs, LPMO a myriad of different enzymes including several H2O2-consuming 27 Kinetic parameters enzymes like different peroxidases and peroxygenases . Com- deduction parative studies of the k /K values of the various H O - S S-O cat mH2O2 2 2 consuming enzymes in such secretomes could provide some Fig. 1 Two enzymes, a reference enzyme (Eref, i.e., SmAA10A or HRP) insight into the relevance of the H2O2-driven LPMO reaction and fl and an LPMO of interest compete for the H O co-substrate. The rate of the ow of H2O2 through different enzyme reactions. In this 2 2 the reference reaction (v ) is a function of the concentration of the LPMO regard, one must bear in mind that the use of kcat/K values as ref mH2O2 k K performance metrics in comparing different enzymes assumes that of interest. Provided with the cat/ m (for H2O2) value for reference reaction one can calculate the kcat/Km (for H2O2) value for the LPMO the concentration of H2O2 is much lower than the apparent K values of the competing enzymes31. reaction (oxidation of substrate, S). Note that, in the case of HRP, the mH2O2 ref Here, inspired by the work of Wang et al.32 who demon- reference substrate (S ) is oxidized but the oxygen atom is not ref strated in 2013 that the fosfomycin-producing epoxidase HppE incorporated into S . 2 NATURE COMMUNICATIONS | (2020) 11:5786 | https://doi.org/10.1038/s41467-020-19561-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19561-8 ARTICLE 33,34 mechanism are given by Eqs. (1) and (2), respectively . absence of competing enzyme). Provided with the value of [E]50 ½ and the values of the parameters of the reference reaction, the d H2O2 v ¼À apparent kcat/K for the enzyme of interest can be calculated ref dt mH2O2 Âà Âà using Eq. (6). kref Eref ½H O Sref ! ! ! ¼ cat 2 2 Âà ÂÃ; app app Âà ref ref ref ref ref ref ref ref K K þ K ½þH O K S þ ½H O S kcat kcat E iS mH2O2 mS 2 2 mH2O2 2 2 ¼ : ð6Þ ref ½ KmH O K E ð1Þ 2 2 mH2O2 50 Âà Âà Finally, the true kcat/K value can be found from the ½ ref ref ½ref mH2O2 d H2O2 kcat E HÂÃ2O2 S Âà analysis of (k /K )app values measured at different [S], v ¼À ¼ : cat mH2O2 ref ref ½þref ref þ ½ref dt KmS H2O2 KmH O S H2O2 S according to Eq.
Recommended publications
  • Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UPCommons. Portal del coneixement obert de la UPC Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution Patricia Molina-Espeja,[a] Marina Cañellas,[b] Francisco J. Plou,[a] Martin Hofrichter,[c] Fatima Lucas,[b] Victor Guallar[b] and Miguel Alcalde*[a] Abstract: There is an increasing interest in enzymes that catalyze hydroxylation) to the non-natural olefin cyclopropanation by the hydroxylation of naphthalene under mild conditions and with carbene transfer. Indeed, these enzymes can transform minimal requirements. To address this challenge, an extracellular naphthalene into 1-naphthol by either harnessing the peroxide fungal aromatic peroxygenase with mono(per)oxygenase activity shunt pathway or through their natural NAD(P)H dependent was engineered to selectively convert naphthalene into 1-naphthol. activity.[12-15] More recently, directed evolution of toluene ortho- Mutant libraries constructed by random mutagenesis and DNA monooxygenase (TOM) with a whole-cell biocatalytic system recombination were screened for peroxygenase activity on was described.[16-18] However, the poor enzyme stability and the naphthalene while quenching the undesired peroxidative activity on reliance on expensive redox cofactors and reductase domains 1-naphthol (one-electron oxidation). The resulting double mutant have precluded the practical application of these enzymes in (G241D-R257K) of this process was characterized biochemically specific industrial settings. and computationally. The conformational changes produced by directed evolution improved the substrate´s catalytic position. Over a decade ago, the first “true natural” aromatic Powered exclusively by catalytic concentrations of H2O2, this soluble peroxygenase was discovered (EC 1.11.2.1; also referred to as and stable biocatalyst has total turnover numbers of 50,000, with unspecific peroxygenase, UPO).[19] This enzyme was recently high regioselectivity (97%) and reduced peroxidative activity.
    [Show full text]
  • Selective Synthesis of the Human Drug Metabolite 5′-Hydroxypropranolol by an Evolved Self-Sufficient Peroxygenase
    This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Research Article Cite This: ACS Catal. 2018, 8, 4789−4799 pubs.acs.org/acscatalysis Selective Synthesis of the Human Drug Metabolite 5′- Hydroxypropranolol by an Evolved Self-Sufficient Peroxygenase † ‡ § § Patricia Gomez de Santos, Marina Cañellas, Florian Tieves, Sabry H. H. Younes, † ∥ § ‡ † Patricia Molina-Espeja, Martin Hofrichter, Frank Hollmann, Victor Guallar, and Miguel Alcalde*, † Department of Biocatalysis, Institute of Catalysis, CSIC, 28049 Madrid, Spain ‡ Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, 08034 Barcelona, Spain § Department of Biotechnology, Delft University of Technology, van der Massweg 9, 2629HZ Delft, The Netherlands ∥ Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Mark 23, 02763 Zittau, Germany *S Supporting Information ABSTRACT: Propranolol is a widely used beta-blocker that is metabolized by human liver P450 monooxygenases into equipotent hydroxylated human drug metabolites (HDMs). It is paramount for the pharmaceutical industry to evaluate the toxicity and activity of these metabolites, but unfortunately, their synthesis has hitherto involved the use of severe conditions, with poor reaction yields and unwanted by- products. Unspecific peroxygenases (UPOs) catalyze the selective oxyfunctionalization of C−H bonds, and they are of particular interest in synthetic organic chemistry. Here, we describe the engineering of UPO from Agrocybe aegerita for the efficient synthesis of 5′-hydroxypropranolol (5′-OHP). We employed a structure-guided evolution approach combined with computational analysis, with the aim of avoiding unwanted phenoxyl radical coupling without having to dope the reaction with radical scavengers.
    [Show full text]
  • Structural Insights Into the Substrate Promiscuity of a Laboratory-Evolved Peroxygenase
    Articles Cite This: ACS Chem. Biol. 2018, 13, 3259−3268 pubs.acs.org/acschemicalbiology Structural Insights into the Substrate Promiscuity of a Laboratory-Evolved Peroxygenase † ∥ ‡ ∥ ‡ Mercedes Ramirez-Escudero, , Patricia Molina-Espeja, , Patricia Gomez de Santos, § † ‡ Martin Hofrichter, Julia Sanz-Aparicio,*, and Miguel Alcalde*, † Department of Crystallography & Structural Biology, Institute of Physical Chemistry “Rocasolano”, CSIC, 28006 Madrid, Spain ‡ Department of Biocatalysis, Institute of Catalysis, CSIC, 28049 Madrid, Spain § Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Mark 23, 02763 Zittau, Germany *S Supporting Information ABSTRACT: Because of their minimal requirements, sub- strate promiscuity and product selectivity, fungal peroxyge- nases are now considered to be the jewel in the crown of C−H oxyfunctionalization biocatalysts. In this work, the crystal struc- ture of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differ- ences were detected between the evolved and native peroxy- genase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Fur- ther mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study
    [Show full text]
  • Europe PMC Funders Group Author Manuscript Nat Catal
    Europe PMC Funders Group Author Manuscript Nat Catal. Author manuscript; available in PMC 2018 May 20. Published in final edited form as: Nat Catal. 2018 January ; 1(1): 55–62. doi:10.1038/s41929-017-0001-5. Europe PMC Funders Author Manuscripts Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalisations Wuyuan Zhang[a], Elena Fernández-Fueyo[a], Yan Ni[a], Morten van Schie[a], Jenö Gacs[a], Rokus Renirie[b], Ron Wever[b], Francesco G. Mutti[b], Dörte Rother[c], Miguel Alcalde[d], and Frank Hollmann[a],* [a]Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands [b]Van’t Hoff Institute for Molecular Sciences (HIMS), Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands [c]Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany [d]Department of Biocatalysis, Institute of Catalysis, CSIC, 28049 Madrid, Spain Abstract Peroxygenases offer attractive means to address challenges in selective oxyfunctionalisation chemistry. Despite their attractiveness, the application of peroxygenases in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant (H2O2). Often atom inefficient peroxide generation systems are required, which show little potential for large Europe PMC Funders Author Manuscripts scale implementation. Here we show that visible light-driven, catalytic water oxidation can be used for in situ generation of H2O2 from water, rendering the peroxygenase catalytically active. In this way the stereoselective oxyfunctionalisation of hydrocarbons can be achieved by simply using the catalytic system, water and visible light.
    [Show full text]
  • Optimization of Photocatalytic Processes: Catalyst Design, Kinetics and Reaction Engineering
    Optimization of Photocatalytic Processes: Catalyst Design, Kinetics and Reaction Engineering Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung der Lehrbefugnis (venia legendi) im Fachgebiet Technische Chemie angenommene kumulative Habilitationsschrift von Dr. rer. nat. Jonathan Zacharias Bloh geboren am 17.09.1983 in Neustadt am Rübenberge, Ortsteil Vesbeck 2021 “On the arid lands there will spring up industrial colonies without smoke and without smokestacks; forests of glass tubes will extend over the plains and glass buildings will rise everywhere; inside of these will take place the photochemical processes that hitherto have been the guarded secret of the plants, but that will have been mastered by human industry which will know how to make them bear even more abundant fruit than nature, for nature is not in a hurry and mankind is. And if in a distant future the supply of coal becomes completely exhausted, civilization will not be checked by that, for life and civilization will continue as long as the sun shines! If our black and nervous civilization, based on coal, shall be followed by a quieter civilization based on the utilization of solar energy, that will not be harmful to progress and to human happiness.” — Giacomo Ciamician, The Photochemistry of the Future, Science, 1912 Abstract The use of light energy to drive chemical reactions has gained increasing interest for a variety of applications in the last decades. This habilitation thesis comprises a selected number of original papers from the author’s own research in this field, particularly concerning semiconductor photocatalysis. Semiconductor photocatalysis has so far been explored for two main applications: The decontamination of air, water or surfaces from unwanted pollutants and the synthesis of value-added chemical products.
    [Show full text]
  • Peroxygenases En Route to Becoming Dream Catalysts
    Delft University of Technology Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? Wang, Yonghua; Lan, Dongming; Durrani, Rabia; Hollmann, Frank DOI 10.1016/j.cbpa.2016.10.007 Publication date 2017 Document Version Final published version Published in Current Opinion in Chemical Biology Citation (APA) Wang, Y., Lan, D., Durrani, R., & Hollmann, F. (2017). Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? Current Opinion in Chemical Biology, 37, 1-9. https://doi.org/10.1016/j.cbpa.2016.10.007 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above. Copyright Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. Available online at www.sciencedirect.com ScienceDirect Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? Yonghua Wang1, Dongming Lan1, Rabia Durrani2 and Frank Hollmann3 Peroxygenases are promising catalysts for preparative classes rely on oxoferryl-heme as the oxygenating species oxyfunctionalization chemistry as they combine the versatility (Compound I) to catalyze a broad range of oxyfunctiona- of P450 monooxygenases with simplicity of cofactor- lization reactions (Scheme 1).
    [Show full text]
  • Journal Pre-Proof
    Journal Pre-proof Advances in enzymatic oxyfunctionalization of aliphatic compounds Carmen Aranda, Juan Carro, Alejandro González-Benjumea, Esteban D. Babot, Andrés Olmedo, Dolores Linde, Angel T. Martínez, Ana Gutiérrez PII: S0734-9750(21)00009-4 DOI: https://doi.org/10.1016/j.biotechadv.2021.107703 Reference: JBA 107703 To appear in: Biotechnology Advances Received date: 7 November 2020 Revised date: 17 January 2021 Accepted date: 25 January 2021 Please cite this article as: C. Aranda, J. Carro, A. González-Benjumea, et al., Advances in enzymatic oxyfunctionalization of aliphatic compounds, Biotechnology Advances (2019), https://doi.org/10.1016/j.biotechadv.2021.107703 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier. Journal Pre-proof Advances in enzymatic oxyfunctionalization of aliphatic compounds Carmen Arandaa¶#, Juan Carrob¶, Alejandro González-Benjumeaa, Esteban D. Babota, Andrés Olmedoa, Dolores Lindeb, Angel T. Martínezb*, Ana Gutiérreza* aInstituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Reina Mercedes 10, 41012 Seville, Spain bCentro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain ¶These two authors equally contributed to the work #Current address: Johnson Matthey, Cambridge Science Park U260, Milton Road, Cambridge CB4 0FP, UK *Corresponding authors: [email protected] (A.
    [Show full text]
  • Modulating Fatty Acid Epoxidation Vs Hydroxylation in a Fungal
    This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Research Article Cite This: ACS Catal. 2019, 9, 6234−6242 pubs.acs.org/acscatalysis Modulating Fatty Acid Epoxidation vs Hydroxylation in a Fungal Peroxygenase † ⊥ ‡ ⊥ † ⊥ # ‡ Juan Carro, , Alejandro Gonzalez-Benjumea,́ , Elena Fernandez-Fueyo,́ , , Carmen Aranda, § ∥ ‡ † Victor Guallar,*, , Ana Gutierrez,́ *, and Angel T. Martínez*, † Centro de Investigaciones Biologicas,́ CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain ‡ Instituto de Recursos Naturales y Agrobiología, CSIC, Reina Mercedes 10, E-41012 Sevilla, Spain § Barcelona Supercomputing Center, Jordi Girona 29, E-08034 Barcelona, Spain ∥ ICREA, Passeig Lluís Companys 23, E-08010 Barcelona, Spain *S Supporting Information ABSTRACT: Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Both enzyme types share the ability to perform selective oxygenation reactions. Moreover, the Marasmius rotula UPO (MroUPO) catalyzes reactions of interest compared with the previously described UPOs, including formation of reactive epoxy fatty acids. To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme channel were predicted using binding and molecular dynamics simulations. Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both the native (wild-type recombinant) MroUPO and the mutated variants were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography−mass spectrometry in combination with kinetic analyses. Interestingly, a small modification of the channel shape in the I153T variant increased the ratio between epoxidized oleic acid and its additionally hydroxylated derivatives.
    [Show full text]
  • Oxyfunctionalization of Alkanes, Alkenes and Alkynes by Unspecific Peroxygenase (EC 1.11.2.1) Oxyfunktionalisierung Von Alkanen
    Oxyfunctionalization of alkanes, alkenes and alkynes by unspecific peroxygenase (EC 1.11.2.1) Oxyfunktionalisierung von Alkanen, Alkenen und Alkinen durch die Unspezifische Peroxygenase (EC 1.11.2.1) Vom Institutsrat des Internationalen Hochschulinstitutes Zittau und der Fakultät Mathematik/ Naturwissenschafften der Technischen Universität Dresden genehmigte Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt von Dipl.-Ing. (Umwelttechnik) Sebastian Peter geboren am 04. Juni 1982 in Erlenbach am Main (Deutschland) Gutachter: Herr Prof. Dr. Martin Hofrichter (TU Dresden, IHI Zittau) Herr Prof. Dr. John T. Groves (Princeton University , USA) Frau Prof. Dr. Katrin Scheibner (Hochschule Lausitz) Tag der Verteidigung: 26.04.2013 Oxyfunctionalization of alkanes, alkenes and alkynes by unspecific peroxygenase (EC 1.11.2.1) Approved by the council of International Graduate School of Zittau and the Faculty of Science of the TU Dresden Academic Dissertation Doctor rerum naturalium (Dr. rer. nat.) by Sebastian Peter, Dipl.-Ing. (Environmental Engeneering) born on June 4, 1982 in Erlenbach am Main (Germany) Reviewer: Prof. Dr. Martin Hofrichter (TU Dresden, IHI Zittau) Prof. Dr. John T. Groves (Princeton University , USA) Prof. Dr. Katrin Scheibner (Hochschule Lausitz) Day of defense: April 26, 2013 CONTENTS Contents CONTENTS ............................................................................................................. I LIST OF ABBREVIATIONS .................................................................................
    [Show full text]
  • A Modular Two Yeast Species Secretion System for the Production and Preparative Application of Unspecific Peroxygenases
    ARTICLE https://doi.org/10.1038/s42003-021-02076-3 OPEN A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases Pascal Püllmann1, Anja Knorrscheidt1, Judith Münch1, Paul R. Palme1, Wolfgang Hoehenwarter1, ✉ Sylvestre Marillonnet1, Miguel Alcalde 2, Bernhard Westermann 1,3 & Martin J. Weissenborn 1,3 Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as 1234567890():,; the oxygen source. However, their heterologous production in a fast-growing organism sui- table for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for pur- ification and split-GFP detection. The modular episomal system is suitable for use in Sac- charomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.
    [Show full text]
  • Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution
    Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution Patricia Molina-Espeja,[a] Marina Cañellas,[b] Francisco J. Plou,[a] Martin Hofrichter,[c] Fatima Lucas,[b] Victor Guallar[b] and Miguel Alcalde*[a] Abstract: There is an increasing interest in enzymes that catalyze hydroxylation) to the non-natural olefin cyclopropanation by the hydroxylation of naphthalene under mild conditions and with carbene transfer. Indeed, these enzymes can transform minimal requirements. To address this challenge, an extracellular naphthalene into 1-naphthol by either harnessing the peroxide fungal aromatic peroxygenase with mono(per)oxygenase activity shunt pathway or through their natural NAD(P)H dependent was engineered to selectively convert naphthalene into 1-naphthol. activity.[12-15] More recently, directed evolution of toluene ortho- Mutant libraries constructed by random mutagenesis and DNA monooxygenase (TOM) with a whole-cell biocatalytic system recombination were screened for peroxygenase activity on was described.[16-18] However, the poor enzyme stability and the naphthalene while quenching the undesired peroxidative activity on reliance on expensive redox cofactors and reductase domains 1-naphthol (one-electron oxidation). The resulting double mutant have precluded the practical application of these enzymes in (G241D-R257K) of this process was characterized biochemically specific industrial settings. and computationally. The conformational changes produced by directed evolution improved the substrate´s catalytic position. Over a decade ago, the first “true natural” aromatic Powered exclusively by catalytic concentrations of H2O2, this soluble peroxygenase was discovered (EC 1.11.2.1; also referred to as and stable biocatalyst has total turnover numbers of 50,000, with unspecific peroxygenase, UPO).[19] This enzyme was recently high regioselectivity (97%) and reduced peroxidative activity.
    [Show full text]
  • Hydroxypropranolol by an Evolved Self-Sufficient Peroxygenase
    This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Research Article Cite This: ACS Catal. 2018, 8, 4789−4799 pubs.acs.org/acscatalysis Selective Synthesis of the Human Drug Metabolite 5′- Hydroxypropranolol by an Evolved Self-Sufficient Peroxygenase † ‡ § § Patricia Gomez de Santos, Marina Cañellas, Florian Tieves, Sabry H. H. Younes, † ∥ § ‡ † Patricia Molina-Espeja, Martin Hofrichter, Frank Hollmann, Victor Guallar, and Miguel Alcalde*, † Department of Biocatalysis, Institute of Catalysis, CSIC, 28049 Madrid, Spain ‡ Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, 08034 Barcelona, Spain § Department of Biotechnology, Delft University of Technology, van der Massweg 9, 2629HZ Delft, The Netherlands ∥ Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Mark 23, 02763 Zittau, Germany *S Supporting Information ABSTRACT: Propranolol is a widely used beta-blocker that is metabolized by human liver P450 monooxygenases into equipotent hydroxylated human drug metabolites (HDMs). It is paramount for the pharmaceutical industry to evaluate the toxicity and activity of these metabolites, but unfortunately, their synthesis has hitherto involved the use of severe conditions, with poor reaction yields and unwanted by- products. Unspecific peroxygenases (UPOs) catalyze the selective oxyfunctionalization of C−H bonds, and they are of particular interest in synthetic organic chemistry. Here, we describe the engineering of UPO from Agrocybe aegerita for the efficient synthesis of 5′-hydroxypropranolol (5′-OHP). We employed a structure-guided evolution approach combined with computational analysis, with the aim of avoiding unwanted phenoxyl radical coupling without having to dope the reaction with radical scavengers.
    [Show full text]