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A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Widersten)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Widersten)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Widersten)
2015 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 18, 2595-2598 p.Article in journal (Refereed) Published
Abstract [en]

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose-6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been presented as a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with these type of bulky acceptor substrates is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here, we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an in vitro evolved aldehyde reductase that reduces the aldehyde product formed after FSA-afforded cleavage of the aldol. The assay is suitable both for steady state enzyme kinetics and real-time activity screening in a 96-well format.

Place, publisher, year, edition, pages
2015. Vol. 16, no 18, 2595-2598 p.
Keyword [en]
aldolase, dehydrogenase, directed evolution, retro-aldol, screening
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-264195DOI: 10.1002/cbic.201500466ISI: 000367720300008PubMedID: 26449620OAI: oai:DiVA.org:uu-264195DiVA: diva2:859479
Funder
Swedish Research Council, 621-2011-6055
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Aldolases for Enzymatic Carboligation: Directed Evolution and Enzyme Structure-Function Relationship Studies
Open this publication in new window or tab >>Aldolases for Enzymatic Carboligation: Directed Evolution and Enzyme Structure-Function Relationship Studies
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA).

Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties.

The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties.  In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties.

The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1318
Keyword
aldolase, 2-deoxyribose-5-phosphate aldolase, fructose-6-phosphate aldolase, directed evolution, enzyme dynamics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-266902 (URN)978-91-554-9411-7 (ISBN)
Public defence
2016-02-05, B22, BMC, Husargatan3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2015-12-07 Created: 2015-11-13 Last updated: 2016-01-13

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Ma, HuanEnugala, Thilak ReddyWidersten, Mikael

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