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Engineering of water networks in class II terpene cyclases underscores the importance of amino acid hydration and entropy in biocatalysis and enzyme design
KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi, Ytbehandlingsteknik. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-1685-4735
Vise andre og tillknytning
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Emneord [en]
Enzyme design, terpene cyclase, hydration, entropy
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-235186OAI: oai:DiVA.org:kth-235186DiVA, id: diva2:1248930
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceTilgjengelig fra: 2018-09-17 Laget: 2018-09-17 Sist oppdatert: 2018-09-18bibliografisk 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

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Kürten, CharlotteUhlén, MathiasSyrén, Per-Olof

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