Systematic Screening for Catalytic Promiscuity in 4-Oxalocrotonate Tautomerase: Enamine Formation and Aldolase Activity

Ellen Zandvoort, Bert-Jan Baas, Wim J. Quax, Gerrit J. Poelarends*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

31 Citations (Scopus)

Abstract

The enzyme 4-oxalocrotonate tautomerase (4-OT) is part of a catabolic pathway for aromatic hydrocarbons in Pseudomonas putida mt-2, where it catalyzes the conversion of 2-hydroxy-2,4-hexadienedioate (1) to 2-oxo-3-hexenedioate (2). 4-OT is a member of the tautomerase superfamily, a group of homologous proteins that are characterized by a beta-alpha-beta structural fold and a catalytic amino-terminal proline. In the mechanism of 4-OT, Pro1 is a general base that abstracts the 2-hydroxyl proton of 1 for delivery to the C-5 position to yield 2. Here, 4-OT was explored for nucleophilic catalysis based on the mechanistic reasoning that its Pro1 residue has the correct protonation state (pK(a) similar to 6.4) to be able to act as a nucleophile at pH 7.3. By using inhibition studies and mass spectrometry experiments it was first demonstrated that 4-OT can use Pro1 as a nucleophile to form an imine/enamine with various aldehyde and ketone compounds. The chemical potential of the smallest enamine (generated from acetaldehyde) was then explored for further reactions by using a small set of selected electrophiles. This systematic screening approach led to the discovery of a new promiscuous activity in wild-type 4-OT: the enzyme catalyzes the aldol condensation of acetaldehyde with benzaldehyde to form cinnamaldehyde. This low-level aldolase activity can be improved 16-fold with a single point mutation (L8R) in 4-OT's active site. The proposed mechanism of the reaction mimicks that used by natural class-I aldolases and designed catalytic aldolase antibodies. An important difference, however, is that these natural and designed aldolases use the primary amine of a lysine residue to form enamines with carbonyl substrates, whereas 4-OT uses the secondary amine of an active-site proline as the nucleophile catalyst. Further systematic screening of 4-OT and related proline-based biocatalysts might prove to be a useful approach to discover new promiscuous carbonyl transformation activities that could be exploited to develop new biocatalysts for carbon-carbon bond formation.

Original languageEnglish
Pages (from-to)602-609
Number of pages8
JournalChemBioChem
Volume12
Issue number4
DOIs
Publication statusPublished - 7-Mar-2011

Keywords

  • aldol reaction
  • catalytic promiscuity
  • enamine catalysis
  • enzyme catalysis
  • tautomerases
  • ALKALINE-PHOSPHATASE
  • ENZYMATIC-ACTIVITIES
  • SUPERFAMILY
  • EVOLUTION
  • ENZYMES
  • MECHANISM
  • RESIDUES
  • ACID
  • DECARBOXYLATION
  • INTERMEDIATE

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