Engineering the promiscuous racemase activity of an arylmalonate decarboxylase

Robert Kourist, Yusuke Miyauchi, Daisuke Uemura, Kenji Miyamoto

Research output: Contribution to journalArticle

21 Citations (Scopus)

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 languageEnglish
Pages (from-to)557-563
Number of pages7
JournalChemistry - A European Journal
Volume17
Issue number2
DOIs
Publication statusPublished - 2011 Jan 10

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Racemases and Epimerases
Substrates
Carboxylic Acids
Carboxylic acids
Bearings (structural)
Ketoprofen
Acidity
Isomers
Catalysis
Reaction rates
Molecular dynamics
Protons
Carbon
Proteins
Atoms
Kinetics
malonate decarboxylase
Computer simulation

Keywords

  • enzymatic promiscuity
  • enzyme catalysis
  • protein engineering
  • racemases

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Engineering the promiscuous racemase activity of an arylmalonate decarboxylase. / Kourist, Robert; Miyauchi, Yusuke; Uemura, Daisuke; Miyamoto, Kenji.

In: Chemistry - A European Journal, Vol. 17, No. 2, 10.01.2011, p. 557-563.

Research output: Contribution to journalArticle

Kourist, Robert ; Miyauchi, Yusuke ; Uemura, Daisuke ; Miyamoto, Kenji. / Engineering the promiscuous racemase activity of an arylmalonate decarboxylase. In: Chemistry - A European Journal. 2011 ; Vol. 17, No. 2. pp. 557-563.
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