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Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
KTH, School of Chemical Science and Engineering (CHE).
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1685-4735
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-4066-2776
2017 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 23, p. 2301-2305Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2017. Vol. 18, no 23, p. 2301-2305
Keywords [en]
biosynthesis, enzyme catalysis, isotope effects, kinetics, reaction mechanisms
National Category
Other Chemical Engineering
Identifiers
URN: urn:nbn:se:kth:diva-220459DOI: 10.1002/cbic.201700443ISI: 000417219500006OAI: oai:DiVA.org:kth-220459DiVA, id: diva2:1170612
Funder
Swedish Research Council, 621-2013-5138AFA Insurance, 17-359Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180104

Available from: 2018-01-04 Created: 2018-01-04 Last updated: 2018-09-13Bibliographically 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

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