Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme design
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1685-4735
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Keywords [en]
Enzyme design, terpene cyclase, hydration, entropy
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-235186OAI: oai:DiVA.org:kth-235186DiVA, id: diva2:1248930
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceAvailable from: 2018-09-17 Created: 2018-09-17 Last updated: 2018-09-18Bibliographically approved
In thesis
1. On Catalytic Mechanisms for Rational Enzyme Design Strategies
Open this publication in new window or tab >>On Catalytic Mechanisms for Rational Enzyme Design Strategies
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2018. p. 113
Series
TRITA-CBH-FOU ; 2018:37
Keywords
catalytic mechanisms, terpene cyclase, triterpene cyclase, solvent dynamics, protein hydration, thermodynamics, quantum tunneling, polycyclization, natural compounds, 𝛼/𝛽-hydrolase, esterase, amidase, enzyme engineering, biocatalysis
National Category
Biocatalysis and Enzyme Technology Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-234940 (URN)978-91-7729-917-2 (ISBN)
Public defence
2018-10-26, K1, Teknikringen 56, KTH main campus, Stockholm, 13:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180914

Available from: 2018-09-18 Created: 2018-09-13 Last updated: 2018-09-19Bibliographically approved

Open Access in DiVA

No full text in DiVA

Search in DiVA

By author/editor
Kürten, CharlotteUhlén, MathiasSyrén, Per-Olof
By organisation
Coating TechnologyScience for Life Laboratory, SciLifeLab
Biochemistry and Molecular BiologyBiocatalysis and Enzyme Technology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 434 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf