Abstract
Variant G74C of arylmalonate decarboxylase (AMDase) from Bordatella bronchoseptica has a unique racemising activity towards profens. By protein engineering, variant G74C/V43A with a 20-fold shift towards promiscuous racemisation was obtained, based on a reduced activity in the decarboxylation reaction and a two-fold increase in the racemisation activity. The mutant showed an extended substrate range, with a 30-fold increase in the reaction rate towards ketoprofen. Molecular dynamics simulations and the substrate profile of the racemase indicate that the steric and polar effects of the substrate structure play a more dominant role on catalysis than mere kinetic α-proton acidity. The observation that the conversion of β,γ-unsaturated carboxylic acids does not lead to a rearrangement to form their α,β isomers indicates a concerted rather than a stepwise mechanism. Interestingly, a substrate bearing a nitro group instead of the carboxylic acid group on the α-carbon atom was also converted by the racemase.
Original language | English |
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Pages (from-to) | 557-63 |
Number of pages | 7 |
Journal | Chemistry - a European Journal |
Volume | 17 |
Issue number | 2 |
DOIs | |
Publication status | Published - 10 Jan 2011 |
Externally published | Yes |
Keywords
- Bordetella bronchiseptica
- Butyrates
- Carboxy-Lyases
- Catalysis
- Computer Simulation
- Genetic Variation
- Models, Molecular
- Molecular Structure
- Mutagenesis, Site-Directed
- Protein Engineering
- Racemases and Epimerases
- Stereoisomerism
- Substrate Specificity
- Journal Article
- Research Support, Non-U.S. Gov't
Fields of Expertise
- Human- & Biotechnology