Engineering the promiscuous racemase activity of an arylmalonate decarboxylase

Robert Kourist, Yusuke Miyauchi, Daisuke Uemura, Kenji Miyamoto

Research output: Contribution to journalArticle

22 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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Keywords

  • enzymatic promiscuity
  • enzyme catalysis
  • protein engineering
  • racemases

ASJC Scopus subject areas

  • Catalysis
  • Organic Chemistry

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