Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
KTH, Skolan för kemivetenskap (CHE).
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-1685-4735
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Skolan för kemivetenskap (CHE), Kemi, Tillämpad fysikalisk kemi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-4066-2776
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. Vol. 18, nr 23, s. 2301-2305
Emneord [en]
biosynthesis, enzyme catalysis, isotope effects, kinetics, reaction mechanisms
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-220459DOI: 10.1002/cbic.201700443ISI: 000417219500006OAI: oai:DiVA.org:kth-220459DiVA, id: diva2:1170612
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
Inngår i avhandling
1. On Catalytic Mechanisms for Rational Enzyme Design Strategies
Åpne denne publikasjonen i ny fane eller vindu >>On Catalytic Mechanisms for Rational Enzyme Design Strategies
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
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:nbn:se:kth:diva-234940 (URN)978-91-7729-917-2 (ISBN)
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

Open Access i DiVA

Fulltekst mangler i DiVA

Andre lenker

Forlagets fulltekst

Søk i DiVA

Av forfatter/redaktør
Eriksson, AdamKürten, CharlotteSyrén, Per-Olof
Av organisasjonen
I samme tidsskrift
ChemBioChem (Print)

Søk utenfor DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric

doi
urn-nbn
Totalt: 164 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf