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Systematic Screening for Catalytic Promiscuity in 4‐Oxalocrotonate Tautomerase: Enamine Formation and Aldolase Activity
Author(s) -
Zandvoort Ellen,
Baas BertJan,
Quax Wim J.,
Poelarends Gerrit J.
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000633
Subject(s) - chemistry , enamine , aldolase a , stereochemistry , nucleophile , active site , sulfolobus solfataricus , ketone , biochemistry , enzyme , organic chemistry , catalysis , archaea , gene
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 β‐α‐β 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 (p K a ∼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.