Bert-Jan Baas

Self-Sufficient Baeyer–Villiger Monooxygenases: Effective Coenzyme Regeneration for Biooxygenation by Fusion Engineering†

Over the past few years, industrial interest in biocatalysts that perform selective oxidative reactions has increased significantly. Baeyer–Villiger monooxygenases (BVMOs) have been identified as a highly versatile class of enzymes for the efficient catalysis of chemo-, regio-, and/or enantioselective oxygenation reactions. Although the most prominent transformation catalyzed by these biocatalysts is a chiral variant of the classical Baeyer–Villiger reaction, the oxygenation of heteroatoms and epoxidation reactions have also been reported. Stoichiometric amounts of O2 and NADPH are required for these reactions. A complication for the largescale application of these reactions is the high cost of the reduced nicotinamide coenzyme. To overcome this problem, several electrochemical and photochemical approaches have been explored. However, the efficiency of these approaches is typically poor. Furthermore, it has been shown that BVMOs require NADP for stability and enantioselective catalysis. An efficient and commonly used method for coenzyme regeneration employs whole cells, especially in combination with the recombinant expression of the required biocatalysts. This strategy has been implemented in BVMOmediated biotransformations with wild-type strains and has proved particularly successful with recombinant overexpression systems. The approach avoids laborious enzyme purification steps and exploits the coenzyme regeneration capacity of the host. Although whole cells have been shown to be effective catalysts for Baeyer–Villiger oxidation, they also exhibit limitations, such as cellular toxicity, enzyme inhibition by the substrate/product, degradation of the product, and poor oxygen-transfer rates. Coenzyme regeneration by using isolated enzymes has also been studied extensively in the past few years. Well-known examples of such NADPH-regenerating enzymes are alcohol dehydrogenase and formate dehydrogenase. A phosphite dehydrogenase (PTDH) was also identified as an effective enzyme for coenzyme regeneration. The favorable thermodynamic equilibrium constant makes the oxidation of phosphite a nearly irreversible process. The exquisite selectivity of PTDH for phosphite also precludes any side reactions, such as those that can occur, for example, when an alcohol dehydrogenase is used. These characteristics make PTDH an ideal candidate for use as a coenzyme regenerating enzyme (CRE) in combination with BVMOs or other NAD(P)H-dependent enzymes. Herein, we report a novel approach to the combination of the catalytic activity of a redox biocatalyst with concomitant coenzyme recycling in a single fusion protein (Scheme 1). During the last decade, a number of fusion protein tags have been developed. These tags are used intensely in life-sciencerelated research and commercial activities. Although the fusion of proteins is a widely applied strategy in, for example, enzyme purification (e.g. the use of glutathione S transferase (GST) tags) and the subcellular visualization of target proteins (e.g. with a green fluorescent protein (GFP) tag), this concept is hardly ever encountered in the context of synthetic applications. Only a few isolated examples in the literature provide evidence that the fusion of separate enzymes can result in improved biocatalytic properties. We report herein on the engineering of a number of representative BVMOs that are linked covalently to soluble NADPH-regenerating phosphite dehydrogenase. This construct enables the use of phosphite as a cheap and sacrificial electron donor with whole cells, cell extracts, and purified enzyme. It was our particular goal to design a self-sufficient two-in-one redox biocatalyst that does not require an additional catalytic entity for coenzyme recycling. As model

Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.

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,

Aqueous Oxidative Heck Reaction as a Protein-Labeling Strategy

An increasing number of chemical reactions are being employed for bio‐orthogonal ligation of detection labels to protein‐bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell lysates. Here we present the palladium‐catalyzed oxidative Heck reaction as a new and robust bio‐orthogonal strategy for linking functionalized arylboronic acids to protein‐bound alkenes in high yields and with excellent chemoselectivity even in the presence of complex protein mixtures from living cells. Advantageously, this reaction proceeds under aerobic conditions, whereas most other metal‐catalyzed reactions require inert atmosphere.

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.

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.

Identification of 6-benzyloxysalicylates as a novel class of inhibitors of 15-lipoxygenase-1

Lipoxygenases metabolize polyunsaturated fatty acids into signalling molecules such as leukotrienes and lipoxins. 15-lipoxygenase-1 (15-LOX-1) is an important mammalian lipoxygenase and plays a crucial regulatory role in several respiratory diseases such as asthma, COPD and chronic bronchitis. Novel potent and selective inhibitors of 15-LOX-1 are required to explore the role of this enzyme in drug discovery. In this study we describe structure activity relationships for 6-benzyloxysalicylates as inhibitors of human 15-LOX-1. Kinetic analysis suggests competitive inhibition and the binding model of these compounds can be rationalized using molecular modelling studies. The most potent derivative 37a shows a Ki value of 1.7 μM. These structure activity relationships provide a basis to design improved inhibitors and to explore 15-LOX-1 as a drug target.

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.