title: Systematic screening for catalytic promiscuity in 4-oxalocrotonate tautomerase: enamine formation and aldolase activity.
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 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 (pK(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.
authors.affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Ellen
authors.initials: E
authors.lastname: Zandvoort
authors.forename: Bert-Jan
authors.initials: BJ
authors.lastname: Baas
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
mesh_terms: D000588:Amines / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D016958:Pseudomonas putida / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D000588:Amines; D005634:Fructose-Bisphosphate Aldolase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases