CARACTERIZACION MULTIESCALAR DE INTERMEDIOS REACTIVOS PARA EL DESCUBRIMIENTO Y DISEÑO DE NUEVAS ACTIVIDADES BIOCATALITICAS

PID2019-111300GA-I00

Nombre agencia financiadora Agencia Estatal de Investigación
Acrónimo agencia financiadora AEI
Programa Programa Estatal de Generación de Conocimiento y Fortalecimiento Científico y Tecnológico del Sistema de I+D+i
Subprograma Subprograma Estatal de Generación de Conocimiento
Convocatoria Proyectos I+D
Año convocatoria 2019
Unidad de gestión Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020
Centro beneficiario UNIVERSITAT DE GIRONA
Identificador persistente http://dx.doi.org/10.13039/501100011033

Publicaciones

Found(s) 9 result(s)
Found(s) 1 page(s)

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

DUGiDocs – Universitat de Girona
  • Acevedo-Rocha, Carlos G.
  • Li, Aitao
  • D’Amore, Lorenzo
  • Hoebenreich, Sabrina
  • Sanchis, Joaquin
  • Lubrano, Paul
  • Ferla, Matteo P.
  • Garcia Borràs, Marc
  • Osuna Oliveras, Sílvia
  • Reetz, Manfred T.
Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s, Support from the Max-Planck-Society and the LOEWE Research cluster SynChemBio is gratefully acknowledged. A.L. thanks the support from the National Key Research and Development Program of China (2019YFA0905000). This study was also supported in part by the European Research Council Horizon 2020 research and innovation programme (ERC-2015-StG-679001, to S.O.), Spanish MINECO (project PGC2018-102192-B-I00, to S.O.; project PID2019-111300GA-I00, to M.G.-B.; and Juan de la Cierva - Incorporación fellowship IJCI-2017-33411, to M.G.-B.), UdG (predoctoral fellowship IFUdG2016, to L.D.), and Generalitat de Catalunya AGAUR (SGR-1707, to S.O.; and Beatriu de Pinós H2020 MSCA-Cofund 2018-BP-00204, to M.G.-B.). M.P.F. is supported by the Wellcome Trust [203141/Z/16/Z] and the NIHR Biomedical Research Centre Oxford




Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

DUGiDocs – Universitat de Girona
  • Knorrscheidt, Anja
  • Soler i Parpal, Jordi
  • Hünecke, Nicole
  • Püllmann, Pascal
  • Garcia Borràs, Marc
  • Weissenborn, Martin J.
Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest, M.J.W., A.K., and N.H. thank the Bundesministerium für
Bildung und Forschung (“Biotechnologie 2020+ Strukturvorhaben:
Leibniz Research Cluster”, 031A360B) for generous
funding. P.P. thanks the Landesgraduiertenförderung Sachsen-
Anhalt for a Ph.D. scholarship. M.G.-B. thanks the Generalitat
de Catalunya AGAUR for a Beatriu de Pinós H2020 MSCACofund
2018-BP-00204 project and the Spanish MICINN
(Ministerio de Ciencia e Innovación) for PID2019-111300GAI00
project, and J.S. thanks the Spanish MIU (Ministerio de
Universidades) for a predoctoral FPU fellowship FPU18/
02380. The computer resources at MinoTauro and the
Barcelona Supercomputing Center BSC-RES are acknowledged
(RES-QSB-2019-3-0009 and RES-QSB-2020-2-0016)




Replication data for: Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

DUGiDocs – Universitat de Girona
  • Knorrscheidt, Anja
  • Soler i Parpal, Jordi
  • Hünecke, Nicole
  • Püllmann, Pascal
  • Garcia Borràs, Marc
  • Weissenborn, Martin J.
Dades associades amb el material complementari (gràfics, taules, figures, ...) de l'article publicat a la revista 'ACS Catalysis', 2021, vol. 11, núm. 12, p.7327-7338. Disponible a https://doi.org/10.1021/acscatal.1c00847, Strategies for primer design, sequences of the utilized UPOs, kinetic plots, GC parameters and original GC chromatograms, activities and selectivities of all tested variants, reaction conditions, calibration curves, and figures of docking studies and MD simulations




Simultaneous screening of multiple substrates with an unspecific peroxygenase enabled modified alkane and alkene oxyfunctionalisations

DUGiDocs – Universitat de Girona
  • Knorrscheidt, Anja
  • Soler i Parpal, Jordi
  • Hünecke, Nicole
  • Püllmann, Pascal
  • Garcia Borràs, Marc
  • Weissenborn, Martin J.
A high throughput GC-MS approach was developed, permitting the simultaneous analysis of up to three substrates and six products quantitatively from one reaction mixture. This screening approach was applied to site-saturation libraries of the novel unspecific peroxygenaseMthUPO. Using this setup enabled substantial insights from a small mutant library. Enzyme variants were identified exhibiting selective alkene epoxidation and substantially shifted regioselectivities to 2- and 1-octanol formations. Computational modelling rationalised the observed selectivity changes, M. J. W, A. K., and N. H. thank the Bundesministerium für Bildung und Forschung (“Biotechnologie 2020+ Strukturvorhaben: Leibniz Research Cluster”, 031A360B) for generous funding. P. P. thanks the Landesgraduiertenförderung Sachsen-Anhalt for a PhD scholarship. The authors thank Eugen Schell for fruitful discussions. M. G. B. thanks the Generalitat de Catalunya AGAUR for a Beatriu de Pinós H2020 MSCA-Cofund 2018-BP-00204 project, the Spanish MICINN (Ministerio de Ciencia e Innovación) for PID2019-111300GA-I00 project, and J. S. thanks the Spanish MIU (Ministerio de Universidades) for a predoctoral FPU fellowship FPU18/02380. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged (RES-QSB-2019-3-262 0009 and RES-QSB-2020-2-0016)




Enzymatic Control over Reactive Intermediates Enables Direct Oxidation of Alkenes to Carbonyls by a P450 Iron-Oxo Species

DUGiDocs – Universitat de Girona
  • Soler i Parpal, Jordi
  • Gergel, Sebastian
  • Klaus, Cindy
  • Hammer, Stephan C.
  • Garcia Borràs, Marc
The aerobic oxidation of alkenes to carbonyls is an important and challenging transformation in synthesis. Recently, a new P450-based enzyme (aMOx) has been evolved in the laboratory to directly oxidize styrenes to their corresponding aldehydes with high activity and selectivity. The enzyme utilizes a heme-based, high-valent iron-oxo species as a catalytic oxidant that normally epoxidizes alkenes, similar to other catalysts. How the evolved aMOx enzyme suppresses the commonly preferred epoxidation and catalyzes direct carbonyl formation is currently not well understood. Here, we combine computational modelling together with mechanistic experiments to study the reaction mechanism and unravel the molecular basis behind the selectivity achieved by aMOx. Our results describe that although both pathways are energetically accessible diverging from a common covalent radical intermediate, intrinsic dynamic effects determine the strong preference for epoxidation. We discovered that aMOx overrides these intrinsic preferences by controlling the accessible conformations of the covalent radical intermediate. This disfavors epoxidation and facilitates the formation of a carbocation intermediate that generates the aldehyde product through a fast 1,2-hydride migration. Electrostatic preorganization of the enzyme active site also contributes to the stabilization of the carbocation intermediate. Computations predicted that the hydride migration is stereoselective due to the enzymatic conformational control over the intermediate species. These predictions were corroborated by experiments using deuterated styrene substrates, which proved that the hydride migration is cis- and enantioselective. Our results demonstrate that directed evolution tailored a highly specific active site that imposes strong steric control over key fleeting biocatalytic intermediates, which is essential for accessing the carbonyl forming pathway and preventing competing epoxidation, This work was supported by the Spanish MICINN (Ministerio
de Ciencia e Innovación) PID2019-111300GA-I00 project and
the Ramón y Cajal program via the RYC 2020-028628-I
fellowship (M.G.B), the Generalitat de Catalunya AGAUR
Beatriu de Pinós H2020 MSCA- COFUND (grant agreement
No 801370) 2018-BP-00204 project (M.G.B.), the Spanish
MIU (Ministerio de Universidades) predoctoral FPU fellowship
FPU18/02380 (J.S.), and the Deutsche Forschungsgemeinschaft via the Emmy Noether fellowship 420112577 (S.C.H.), Open Access funding provided thanks to the CRUE-CSIC agreement with ACS




Changes in Protonation States of In-Pathway Residues can Alter Ligand Binding Pathways Obtained From Spontaneous Binding Molecular Dynamics Simulations

DUGiDocs – Universitat de Girona
  • Giramé, Helena
  • Garcia Borràs, Marc
  • Feixas Geronès, Ferran
Protein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 Å away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations, This work was supported by the Spanish MICINN projects PID 2019-111300GA-I00 (MG-B), RTI 2018-101032-J100 (FF). We thank Spanish MICINN for Ramón y Cajal fellowships RYC 2020-028628-I (MG-B) and RYC 2020-029552-I (FF). We thank the Generalitat de Catalunya for the emerging group CompBioLab (2017 SGR-1707), the consolidated group DiMoCat (2017 SGR-39), for predoctoral FI fellowship 2022 FI_B 00615 (HG) and Beatriu de Pinós H2020 MSCA-Cofund 2018-BP-00204 project (to MG-B)




Regioselective access to orthogonal Diels-Alder C60 bis-adducts and tris-heteroadducts via supramolecular mask strategy

DUGiDocs – Universitat de Girona
  • Pujals, Míriam
  • Pèlachs, Tània
  • Fuertes Espinosa, Carles
  • Parella Coll, Teodor
  • Garcia Borràs, Marc
  • Ribas Salamaña, Xavi
The regioselective polyfunctionalization of highly symmetric spherical Ih-C60 is extremely challenging and usually leads to the formation of regioisomeric mixtures not amenable for high-pressure liquid chromatography (HPLC) purification. Here, we pioneer the use of tetragonal prismatic nanocapsules to perform selective Diels-Alder (DA) functionalization of encapsulated Ih-C60 using acenes. The supramolecular mask allows the regioselective synthesis of either e,e-bis-anthracene-C60 (functionalization at 90°) or the synthesis of trans-1-bis-pentacene-C60 (functionalization at 180°) by changing only the acene length. Moreover, the mask strategy allows one to obtain unprecedented equatorial hetero-tris-functionalized-C60 adducts combining Diels-Alder with Bingel mask regiofunctionalization. Computational modeling provides crucial insights to rationalize the regioselective control exerted by the supramolecular mask on the successive DA cycloadditions. Molecular dynamics (MD) simulations revealed significant differences in the host-guest interactions and equilibrium established between the first-formed anthracene- and pentacene-based mono-adducts with the nanocapsule, which finally determine the observed orthogonal regioselectivity, This work was supported by grants from MINECO-Spain (PID2019-104498GB-I00 to X.R., PID2019-111300GA-I00 and RYC2020-028628-I to M.G.-B., and PGC2018-095808-B-I00 to T.P.), Fundación Areces (RegioSolar project to X.R.), and Generalitat de Catalunya AGAUR (2017SGR264 to X.R. and H2020 MSCA-Cofund Beatriu de Pinós grant 2018-BP-00204 to M.G.-B)




Reversing the Enantioselectivity of Enzymatic Carbene N−H Insertion Through Mechanism-Guided Protein Engineering

DUGiDocs – Universitat de Girona
  • Calvó-Tusell, Carla
  • Liu, Zhen
  • Chen, Kai
  • Arnold, Frances H.
  • Garcia Borràs, Marc
We report a computationally driven approach to access enantiodivergent enzymatic carbene N−H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N−H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er), Research Funding: Generalitat de Catalunya. Grant Number: 2021SGR00623; Ministerio de Ciencia e Innovación. Grant Numbers: PID2019-111300GA-I00, RYC2020-028628-I; NSF Division of Molecular and Cellular Biosciences. Grant Number: 2016137.
Open Access funding provided thanks to the CRUE-CSIC agreement with Wiley




Computational-Aided Engineering of a Selective Unspecific Peroxygenase toward Enantiodivergent β-Ionone Hydroxylation

DUGiDocs – Universitat de Girona
  • Münch, Judith
  • Soler i Parpal, Jordi
  • Hünecke, Nicole
  • Homann, Dominik
  • Garcia Borràs, Marc
  • Weissenborn, Martin J.
Unspecific peroxygenases (UPOs) perform oxyfunctionalizations for a wide range of substrates utilizing H2O2 without the need for further reductive equivalents or electron transfer chains. Tailoring these promising enzymes toward industrial application was intensely pursued in the last decade with engineering campaigns addressing the heterologous expression, activity, stability, and improvements in chemo- and regioselectivity. One hitherto missing integral part was the targeted engineering of enantioselectivity for specific substrates with poor starting enantioselectivity. In this work, we present the engineering of the short-type MthUPO toward the enantiodivergent hydroxylation of the terpene model substrate, β-ionone. Guided by computational modeling, we designed a small smart library and screened it with a GC–MS setup. After two rounds of iterative protein evolution, the activity increased up to 17-fold and reached a regioselectivity of up to 99.6% for the 4-hydroxy-β-ionone. Enantiodivergent variants were identified with enantiomeric ratios of 96.6:3.4 (R) and 0.3:99.7 (S), respectively, J.M. thanks the Friedrich-Naumann-Stiftung für die Freiheit for a PhD scholarship. D.H. thanks the Friedrich-Ebert Stiftung for a PhD scholarship. M.J.W. and J.M. thank the Bundesministerium für Bildung und Forschung (Maßgeschneiderte Inhaltsstoffe 2, 031B0834A) and M.J.W., J.M., and D.H. thank the German Research Foundation (DFG, project ID 43649874, TP A05, RTG 2670) for generous funding. M.G.B. thanks the Spanish MICINN (Ministerio de Ciencia e Innovación) for PID2019-111300GA-I00 project and the Ramón y Cajal program via the RYC 2020-028628-I fellowship. J.S. thanks the Spanish MIU (Ministerio de Universidades) for a predoctoral FPU fellowship FPU18/02380