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Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-1685-4735
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-4066-2776
2016 (English)In: CATALYSTS, ISSN 2073-4344, Vol. 6, no 6, article id 90Article in journal (Refereed) Published
Resource type
Text
Abstract [en]

The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the alpha/beta phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

Place, publisher, year, edition, pages
MDPI AG , 2016. Vol. 6, no 6, article id 90
Keywords [en]
enzyme promiscuity, enzyme catalysis, biocatalysis, reaction mechanisms, molecular modeling, amidase, esterase
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-189936DOI: 10.3390/catal6060090ISI: 000378839100015Scopus ID: 2-s2.0-84975302338OAI: oai:DiVA.org:kth-189936DiVA, id: diva2:950210
Note

QC 20160728

Available from: 2016-07-28 Created: 2016-07-25 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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