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MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry.
Department of Biological Sciences, Brock University, Ontario, Canada.
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1685-4735
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-4066-2776
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2017 (English)In: ACS Omega, ISSN 2470-1343, Vol. 2, no 11, p. 8495-8506Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017. Vol. 2, no 11, p. 8495-8506
National Category
Biocatalysis and Enzyme Technology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-234939DOI: 10.1021/acsomega.7b01084ISI: 000418744100113OAI: oai:DiVA.org:kth-234939DiVA, id: diva2:1247980
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180914

Available from: 2018-09-13 Created: 2018-09-13 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

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Publisher's full texthttps://pubs.acs.org/doi/abs/10.1021/acsomega.7b01084

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