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Updated: May 15, 2026
10:21
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
New structural motif for carboxylic acid perhydrolases
DeLu Tyler Yin1, Vince M Purpero, Ryota Fujii
1University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics, and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108, USA.
Chemistry (Weinheim an Der Bergstrasse, Germany)
|January 18, 2013
Summary
Modifying the oxyanion loop of Pseudomonas fluorescens esterase (PFE) significantly enhances its perhydrolysis activity. Specific mutations, like L29I, boost peroxycarboxylic acid production, revealing new catalytic mechanisms.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Serine hydrolases can perform promiscuous perhydrolysis, converting carboxylic acids to peroxycarboxylic acids.
- Previous studies identified a proline residue in the oxyanion loop of carboxylic acid perhydrolases.
Purpose of the Study:
- To investigate the role of the proline residue in the oxyanion loop of Pseudomonas fluorescens esterase (PFE) for perhydrolysis activity.
- To identify other mutations that enhance perhydrolysis in PFE.
Main Methods:
- Site-directed mutagenesis of PFE at the L29 position.
- Enzyme kinetics assays to measure perhydrolysis rates and selectivity.
- X-ray crystallography to determine the structure of PFE variants.
- Molecular modeling to elucidate the reaction mechanism.
Main Results:
- The L29P variant of PFE showed a 43-fold increase in perhydrolysis activity compared to wild type.
- Saturation mutagenesis identified six additional substitutions enhancing perhydrolysis, with L29I PFE being 83-fold faster than wild type.
- L29I PFE exhibited similar selectivity for hydrogen peroxide and fast acetyl-enzyme formation as L29P PFE.
- X-ray structures of L29I PFE revealed two oxyanion loop conformations, one creating a binding site for a second acetate.
Conclusions:
- The proline in the oxyanion loop is not essential for high perhydrolysis activity; other substitutions can also significantly enhance it.
- The L29I mutation in PFE leads to a novel catalytic mechanism involving an additional acetate binding site and hydrogen bonding.
- Understanding these structural and mechanistic insights can guide the engineering of esterases for improved peroxycarboxylic acid production.
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