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On Catalytic Mechanisms for Rational Enzyme Design Strategies
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-1685-4735
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Enzymes enable life by promoting chemical reactions that govern the metabolism of all living organisms. As green catalysts, they have been extensively used in industry. However, to reach their full potential, engineering is often required, which can benefit from a detailed understanding of the underlying reaction mechanism.

In Paper I, we screened for an esterase with promiscuous amidase activity capitalizing on a key hydrogen bond acceptor that is able to stabilize the rate limiting nitrogen inversion. In silicoanalyses revealed the esterase patatin as promising target that indeed catalyzed amide hydrolysis when tested in vitro. While key transition state stabilizers for amide hydrolysis are known, we were interested in increasing our fundamental understanding of terpene cyclase catalysis (Paper II-V). In Paper II, kinetic studies in D2O-enriched buffers using a soluble diterpene cyclase suggested that hydrogen tunneling is part of the rate-limiting protonation step. In Paper III, we performed intense computational analyses on a bacterial triterpene cyclase to show the influence of water flow on catalysis. Water movement in the active site and in specific water channels, influencing transition state formation, was detected using streamline analysis. In Paper IV and V, we focused on the human membrane-bound triterpene cyclase oxidosqualene cyclase. We first established a bacterial expression and purification protocol in Paper IV, before performing detailed in vitroand in silicoanalyses in Paper V. Our analyses showed an entropy-driven reaction mechanism and the existence of a tunnel network in the structure of the human enzyme. The influence of water network rearrangements on the thermodynamics of the transition state formation were confirmed. Introducing mutations in the tunnel lining residues severely affected the temperature dependence of the reaction by changing the water flow and network rearrangements in the tunnels and concomitant the active site.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2018. , s. 113
Serie
TRITA-CBH-FOU ; 2018:37
Emneord [en]
catalytic mechanisms, terpene cyclase, triterpene cyclase, solvent dynamics, protein hydration, thermodynamics, quantum tunneling, polycyclization, natural compounds, 𝛼/𝛽-hydrolase, esterase, amidase, enzyme engineering, biocatalysis
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-234940ISBN: 978-91-7729-917-2 (tryckt)OAI: oai:DiVA.org:kth-234940DiVA, id: diva2:1247991
Disputas
2018-10-26, K1, Teknikringen 56, KTH main campus, Stockholm, 13:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20180914

Tilgjengelig fra: 2018-09-18 Laget: 2018-09-13 Sist oppdatert: 2018-09-19bibliografisk kontrollert
Delarbeid
1. Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity
Åpne denne publikasjonen i ny fane eller vindu >>Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity
2016 (engelsk)Inngår i: CATALYSTS, ISSN 2073-4344, Vol. 6, nr 6, artikkel-id 90Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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

sted, utgiver, år, opplag, sider
MDPI AG, 2016
Emneord
enzyme promiscuity, enzyme catalysis, biocatalysis, reaction mechanisms, molecular modeling, amidase, esterase
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-189936 (URN)10.3390/catal6060090 (DOI)000378839100015 ()2-s2.0-84975302338 (Scopus ID)
Merknad

QC 20160728

Tilgjengelig fra: 2016-07-28 Laget: 2016-07-25 Sist oppdatert: 2018-09-18bibliografisk kontrollert
2. Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
Åpne denne publikasjonen i ny fane eller vindu >>Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
2017 (engelsk)Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, nr 23, s. 2301-2305Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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

sted, utgiver, år, opplag, sider
Wiley-VCH Verlagsgesellschaft, 2017
Emneord
biosynthesis, enzyme catalysis, isotope effects, kinetics, reaction mechanisms
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-220459 (URN)10.1002/cbic.201700443 (DOI)000417219500006 ()
Forskningsfinansiär
Swedish Research Council, 621-2013-5138AFA Insurance, 17-359Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20180104

Tilgjengelig fra: 2018-01-04 Laget: 2018-01-04 Sist oppdatert: 2018-09-13bibliografisk kontrollert
3. MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
Åpne denne publikasjonen i ny fane eller vindu >>MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
Vise andre…
2017 (engelsk)Inngår i: ACS Omega, ISSN 2470-1343, Vol. 2, nr 11, s. 8495-8506Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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

sted, utgiver, år, opplag, sider
American Chemical Society (ACS), 2017
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-234939 (URN)10.1021/acsomega.7b01084 (DOI)000418744100113 ()
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20180914

Tilgjengelig fra: 2018-09-13 Laget: 2018-09-13 Sist oppdatert: 2018-09-18bibliografisk kontrollert
4. Overexpression of functional human oxidosqualene cyclase in Escherichia coli
Åpne denne publikasjonen i ny fane eller vindu >>Overexpression of functional human oxidosqualene cyclase in Escherichia coli
2015 (engelsk)Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 115, s. 46-53Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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

sted, utgiver, år, opplag, sider
Elsevier, 2015
Emneord
E. coli, Expression and purification, Membrane protein, Oxidosqualene cyclase, Triterpene cyclase
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-176183 (URN)10.1016/j.pep.2015.04.015 (DOI)2-s2.0-84941878054 (Scopus ID)
Forskningsfinansiär
Swedish Research Council, 621-2013-5138
Merknad

QC 20151125

Tilgjengelig fra: 2015-11-25 Laget: 2015-11-02 Sist oppdatert: 2018-09-13bibliografisk kontrollert
5. Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme design
Åpne denne publikasjonen i ny fane eller vindu >>Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme design
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Emneord
Enzyme design, terpene cyclase, hydration, entropy
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-235186 (URN)
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Tilgjengelig fra: 2018-09-17 Laget: 2018-09-17 Sist oppdatert: 2018-09-18bibliografisk kontrollert

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