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Overexpression of functional human oxidosqualene cyclase in Escherichia coli
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-1685-4735
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-4066-2776
2015 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 115, p. 46-53Article in journal (Refereed) Published
Abstract [en]

The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7 mg protein/g cells (hOSC in Pichia pastoris) after 48 h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9 mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.

Place, publisher, year, edition, pages
Elsevier, 2015. Vol. 115, p. 46-53
Keywords [en]
E. coli, Expression and purification, Membrane protein, Oxidosqualene cyclase, Triterpene cyclase
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-176183DOI: 10.1016/j.pep.2015.04.015Scopus ID: 2-s2.0-84941878054OAI: oai:DiVA.org:kth-176183DiVA, id: diva2:874008
Funder
Swedish Research Council, 621-2013-5138
Note

QC 20151125

Available from: 2015-11-25 Created: 2015-11-02 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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Kürten, CharlotteUhlén, MathiasSyrén, Per-Olof

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