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Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
2014 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 10, 2387-2398 p.Article in journal (Refereed) Published
Abstract [en]

We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π–π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

Place, publisher, year, edition, pages
2014. Vol. 281, no 10, 2387-2398 p.
National Category
Other Chemistry Topics
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-207476DOI: 10.1111/febs.12791ISI: 000336451900007OAI: oai:DiVA.org:uu-207476DiVA: diva2:648328
Available from: 2013-09-15 Created: 2013-09-15 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Oxidation of 1,2-Diols Using Alcohol Dehydrogenases: From Kinetic Characterization to Directed Evolution
Open this publication in new window or tab >>Oxidation of 1,2-Diols Using Alcohol Dehydrogenases: From Kinetic Characterization to Directed Evolution
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes as catalysts for chemical transformations has emerged as a “greener” alternative to traditional organic synthesis. An issue to solve though, is that enzymes are designed by nature to catalyze reactions in a living cell and therefore, in many cases, do not meet the requirements of a suitable biocatalyst. By mimicking Darwinian evolution these problems can be addressed in vitro by different types of directed evolution strategies.

α-Hydroxy aldehydes and α-hydroxy ketones are important building blocks in the synthesis of natural products, fine chemicals and pharmaceuticals. In this thesis, two alcohol dehydrogenases, FucO and ADH-A, have been studied. Their potentials to serve as useful biocatalysts for the production of these classes of molecules have been investigated, and shown to be good. FucO for its strict regiospecificity towards primary alcohols and that it strongly prefers the S-enantiomer of diol substrates. ADH-A for its regiospecificity towards secondary alcohols, its enantioselectivity and that is has the ability to use a wide variety of bulky substrates. The kinetic mechanisms of these enzymes were investigated using pre-steady state kinetics, product inhibition, kinetic isotope effects and solvent viscosity effects, and in both cases, the rate limiting steps were pin-pointed to conformational changes occurring at the enzyme-nucleotide complex state. These characterizations provide an important foundation for further studies on these two enzymes.  

FucO is specialized for activity with small aliphatic substrates but is virtually inactive with aryl-substituted compounds. By the use of iterative saturation mutagenesis, FucO was re-engineered and several enzyme variants active with S-3-phenylpropane-1,2-diol and phenylacetaldehyde were obtained. It was shown that these variants capability to act on larger substrates are mainly due to an enlargement of the active site cavity. Furthermore, several amino acids which are important for catalysis and specificity were identified. Phe254 interacts with aryl-substituted substrates through π-π stacking and may be essential for activity with these larger substrates. One mutation caused a loss in the interactions made between the enzyme and the nucleotide and thereby enhanced the turnover number for the preferred substrate

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1081
Keyword
enzyme kinetics, alcohol dehydrogenase, directed evolution, enzyme engineering, diol, α-hydroxy aldehyde
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-208139 (URN)978-91-554-8763-8 (ISBN)
Public defence
2013-11-08, B42, Husargatan 3, BMC, Uppsala universitet, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-10-18 Created: 2013-09-24 Last updated: 2014-01-23Bibliographically approved

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Blikstad, CeciliaWidersten, Mikael

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