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 language | English |
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Pages (from-to) | 602-609 |
Number of pages | 8 |
Journal | ChemBioChem |
Volume | 12 |
Issue number | 4 |
DOIs | |
Publication status | Published - 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