In recent years, organocatalysis has become one of the main areas in asymmetric catalysis of carbon-carbon bond-forming reactions.[1] The fast evolution of the organocatalysis field has been particularly fueled by aminocatalysis, in which secondary and primary amines react with carbonyl compounds to give enamine and iminium ion intermediates. The field was completely transformed during the last two decades by the seminal contributions of List, [2] MacMillan, [3] Yamaguchi, [4] and co-workers. The natural chiral amino acid proline and derivatives thereof were found to be powerful organocatalysts. These secondary amines are applied in substoichiometric quantities and afford high product yields and enantioselectivities in fundamental carbon-carbon bond-forming reactions such as aldolizations, [1, 2, 3b] Michael additions, [1, 4,5] Mannich reactions, [1,6] and Knoevenagel condensations. [1,7] Inspired by the versatile success of proline and its derivatives as organocatalysts, we examined whether the enzyme 4-oxalocrotonate tautomerase (4-OT), [8] which carries a catalytic amino-terminal proline (Pro = P), might be suitable to promiscuously catalyze carbon-carbon bondforming reactions. Herein, we describe the discovery and characterization of two 4-OT-catalyzed asymmetric carboncarbon bond-forming Michael-type addition reactions. Considering our reported 4-OT-catalyzed aldolizations, [9] this work is a pivotal step forward towards our aim to bridge organocatalysis and biocatalysis by developing a new class of biocatalysts that use the powerful proline-based enamine mechanism of organocatalysts [1] but that take advantage of the water solubility and relatively high catalytic rates available with enzymes. A few elegant studies on promiscuous enzyme-catalyzed carbon-carbon bond-forming Michael additions have been reported, but most of these reactions proceed in organic solvents and with moderate stereocontrol.

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.

Call me Michaelase: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to a collection of aromatic and aliphatic nitroolefins with high stereoselectivity producing precursors of γ-aminobutyric acid (GABA) analogues.

Abstract:The substrate scope of the flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2), recombinantly produced in Escherichia coli, was explored. While it has been established that AldO efficiently oxidizes alditols to d-aldoses, this study revealed that the enzyme is also active with a broad range of aliphatic and aromatic alcohols. Alcohols containing hydroxy groups at the C-1 and C-2 positions like 1,2,4-butanetriol (K m = 170 mM, k cat = 4.4 s À1 ), 1,2-pentanediol (K m = 52 mM, k cat = 0.85 s À1) and 1,2-hexanediol (K m = 97 mM, k cat = 2.0 s À1 ) were readily accepted by AldO. Furthermore, the enzyme was highly enantioselective for the oxidation of 1,2-diols [e.g., for 1-phenyl-1,2-ethanediol the (R)-enantiomer was preferred with an E-value of 74]. For several diols the oxidation products were determined by GC-MS and NMR. Interestingly, for all tested 1,2-diols the products were found to be the a-hydroxy acids instead of the expected a-hydroxy aldehydes. Incubation of (R)-1-phenyl-1,2-ethanediol with O) revealed that a second enzymatic oxidation step occurs via the hydrate product intermediate. The relaxed substrate specificity, excellent enantioselectivity, and independence of coenzymes make AldO an attractive enzyme for the preparation of optically pure 1,2-diols and a-hydroxy acids.

Double play: The enzyme 4-oxalocrotonate tautomerase (4-OT) catalyzes not only the initial cross-coupling of acetaldehyde and benzaldehyde to yield 3-hydroxy-3-phenylpropanal, but also the subsequent dehydration of this aldol compound to yield cinnamaldehyde as the final product. Mechanism-inspired engineering provided an active site mutant (F50A) with strongly enhanced aldol condensation activity.