Ellen Zandvoort

Bridging between Organocatalysis and Biocatalysis: Asymmetric Addition of Acetaldehyde to β‐Nitrostyrenes Catalyzed by a Promiscuous Proline‐Based Tautomerase

In recent years, organocatalysis has become one of the main areas in asymmetric catalysis of carbon–carbon bond-forming reactions. 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, MacMillan, Yamaguchi, 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, 2,3b] Michael additions, 4, 5] Mannich reactions, 6] and Knoevenagel condensations. Inspired by the versatile success of proline and its derivatives as organocatalysts, we examined whether the enzyme 4-oxalocrotonate tautomerase (4-OT), 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 carbon– carbon bond-forming Michael-type addition reactions. Considering our reported 4-OT-catalyzed aldolizations, 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 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. 4-OT is a stable enzyme composed of six identical subunits of only 62 amino acid residues each. It belongs to the tautomerase superfamily, a group of homologous proteins that share a conserved catalytic amino-terminal proline and a characteristic b-a-b structural fold. 12] 4-OT takes part in a degradation pathway for aromatic hydrocarbons in Pseudomonas putida mt-2, where it catalyzes the tautomerization of 2-hydroxy-2,4-hexadienedioate (1) into 2-oxo-3-hexenedioate (2, Scheme 1). The key catalytic residues of 4-OTare Pro-1,

Biocatalytic Michael-Type Additions of Acetaldehyde to Nitroolefins with the Proline-Based Enzyme 4-Oxalocrotonate Tautomerase Yielding Enantioenriched γ-Nitroaldehydes

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.

Promiscuous Catalysis of Asymmetric Michael-Type Additions of Linear Aldehydes to β-Nitrostyrene by the Proline-Based Enzyme 4-Oxalocrotonate Tautomerase

Exploiting catalytic promiscuity: The proline-based enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes asymmetric Michael-type additions of linear aldehydes--ranging from acetaldehyde to octanal--to trans-β-nitrostyrene in aqueous solvent. The presence of 1.4 mol% of 4-OT effected formation of the anticipated γ-nitroaldehydes in fair to good yields with dr values of up to 93:7 and ee values of up to 81 %.

Recent Advances in the Study of Enzyme Promiscuity in the Tautomerase Superfamily

Catalytic promiscuity and evolution: Many enzymes exhibit catalytic promiscuity--the ability to catalyze reactions other than their biologically relevant one. These reactions can serve as starting points for both natural and laboratory evolution of new enzymatic functions. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily are discussed.

Systematic screening for catalytic promiscuity in 4-oxalocrotonate tautomerase: enamine formation and aldolase activity.

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 (pKa∼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‐OTs 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.

Enhancement of the Promiscuous Aldolase and Dehydration Activities of 4-Oxalocrotonate Tautomerase by Protein Engineering

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.

Structural and Functional Characterization of a Macrophage Migration Inhibitory Factor Homologue from the Marine Cyanobacterium Prochlorococcus marinus

Macrophage migration inhibitory factor (MIF) is a multifunctional mammalian cytokine, which exhibits tautomerase and oxidoreductase activity. MIF homologues with pairwise sequence identities to human MIF ranging from 31% to 41% have been detected in various cyanobacteria. The gene encoding the MIF homologue from the marine cyanobacterium Prochlorococcus marinus strain MIT9313 has been cloned and the corresponding protein (PmMIF) overproduced, purified, and subjected to functional and structural characterization. Kinetic and (1)H NMR spectroscopic studies show that PmMIF tautomerizes phenylenolpyruvate and (p-hydroxyphenyl)enolpyruvate at low levels. The N-terminal proline of PmMIF is critical for these reactions because the P1A mutant has strongly reduced tautomerase activities. PmMIF shows high structural homology with mammalian MIFs as revealed by a crystal structure of PmMIF at 1.63 A resolution. MIF contains a Cys-X-X-Cys motif that mediates oxidoreductase activity, which is lacking from PmMIF. Engineering of the motif into PmMIF did not result in oxidoreductase activity but increased the tautomerase activity 8-fold. The shared tautomerase activities and the conservation of the beta-alpha-beta structural fold and key functional groups suggest that eukaryotic MIFs and cyanobacterial PmMIF are related by divergent evolution from a common ancestor. While several MIF homologues have been identified in eukaryotic parasites, where they are thought to play a role in modulating the host immune response, PmMIF is the first nonparasitic, bacterial MIF-like protein characterized in detail. This work sets the stage for future studies which could address the question whether a MIF-like protein from a free-living bacterium possesses immunostimulatory features similar to those of mammalian MIFs and MIF-like proteins found in parasitic nematodes and protozoa.

Characterization of a newly identified mycobacterial tautomerase with promiscuous dehalogenase and hydratase activities reveals a functional link to a recently diverged cis-3-chloroacrylic acid dehalogenase.

The enzyme cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is found in a bacterial pathway that degrades a synthetic nematocide, cis-1,3-dichloropropene, introduced in the 20th century. The previously determined crystal structure of cis-CaaD and its promiscuous phenylpyruvate tautomerase (PPT) activity link this dehalogenase to the tautomerase superfamily, a group of homologous proteins that are characterized by a catalytic amino-terminal proline and a β-α-β structural fold. The low-level PPT activity of cis-CaaD, which may be a vestige of the function of its progenitor, prompted us to search the databases for a homologue of cis-CaaD that was annotated as a putative tautomerase and test both its PPT and cis-CaaD activity. We identified a mycobacterial cis-CaaD homologue (designated MsCCH2) that shares key sequence and active site features with cis-CaaD. Kinetic and 1H NMR spectroscopic studies show that MsCCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-chloroacrylic acid. To further probe the active site of MsCCH2, the enzyme was incubated with 2-oxo-3-pentynoate (2-OP). At pH 8.5, MsCCH2 is inactivated by 2-OP due to the covalent modification of Pro-1, suggesting that Pro-1 functions as a nucleophile at pH 8.5 and attacks 2-OP in a Michael-type reaction. At pH 6.5, however, MsCCH2 exhibits hydratase activity and converts 2-OP to acetopyruvate, which implies that Pro-1 is cationic at pH 6.5 and not functioning as a nucleophile. At pH 7.5, the hydratase and inactivation reactions occur simultaneously. From these results, it can be inferred that Pro-1 of MsCCH2 has a pKa value that lies in between that of a typical tautomerase (pKa of Pro-1∼6) and that of cis-CaaD (pKa of Pro-1∼9). The shared activities and structural features, coupled with the intermediate pKa of Pro-1, suggest that MsCCH2 could be characteristic of an evolutionary intermediate along the past route for the divergence of cis-CaaD from an unknown superfamily tautomerase. This makes MsCCH2 an ideal candidate for laboratory evolution of its promiscuous dehalogenase activity, which could identify additional features necessary for a fully active cis-CaaD. Such results will provide insight into pathways that could lead to the rapid divergent evolution of an efficient cis-CaaD enzyme.

An Unexpected Promiscuous Activity of 4-Oxalocrotonate Tautomerase : The cis-trans Isomerisation of Nitrostyrene

Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (k(cat) /K(m) = 1.9×10(3)  M(-1)  s(-1) ).

Dehalogenation of an Anthropogenic Compound by an Engineered Variant of the Mouse Cytokine Macrophage Migration Inhibitory Factor

An unconventional dehalogenase: An engineered variant (I64V/V106L) of the mouse cytokine macrophage migration inhibitory factor (MIF) promiscuously catalyzes the hydrolytic dehalogenation of the xenobiotic organohalogen trans-3-chloroacrylic acid to acetaldehyde. Although the dehalogenase activity of this MIF variant is quite low, it achieves an ~10(9) -fold rate enhancement, matching those of conventional enzymes acting on their natural substrates.