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The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple β-α-β structural scaffold. Most members are constructed from a single-core β-α-β motif or two consecutively fused β-α-β motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

A Pseudomonas sp. UW4 protein (UniProt K9NIA5) of unknown function was identified as similar to 4-oxalocrotonate tautomerase (4-OT)-like and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)-like subgroups of the tautomerase superfamily (TSF). This protein lacks only Tyr-103 of the amino acids critical for cis-CaaD activity (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114). As it may represent an important variant of these enzymes, its kinetic and structural properties have been determined. The protein shows tautomerase activity with phenylenolpyruvate, but lacks native 4-OT activity and dehalogenase activity with the isomers of 3-chloroacrylic acid. It shows mostly low-level hydratase activity at pH 7.0, converting 2-oxo-3-pentynoate to acetopyruvate, consistent with cis-CaaD-like behavior. At pH 9.0, this compound results primarily in covalent modification of Pro-1, which is consistent with 4-OT-like behavior. These observations could reflect a pKa for Pro-1 that is closer to that of cis-CaaD (∼9.2) than to 4-OT (∼6.4). A structure of the native enzyme, at 2.6 A resolution, highlights differences at the active site from those of 4-OT and cis-CaaD that add to our understanding of how contemporary TSF reactions and mechanisms may have diverged from a common 4-OT-like ancestor.

The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure-function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis-3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase-like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies.

A 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata, and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo- and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF). The TSF has two distinct features: members are composed of a single β-α-β unit (homo- and heterohexamer) or two consecutively joined β-α-β units (trimer) and generally have a catalytic amino-terminal proline. The enzyme, designated as fused 4-OT, functions as a 4-OT where the active site groups (Pro-1, Arg-39, Arg-76, Phe-115, Arg-127) mirror those in the canonical 4-OT from Pseudomonas putida mt-2. Inactivation by 2-oxo-3-pentynoate suggests that Pro-1 of fused 4-OT has a low p Ka enabling the prolyl nitrogen to function as a general base. A remarkable feature of the fused 4-OT is the absence of P3 rotational symmetry in the structure (1.5 A resolution). The asymmetric arrangement of the trimer is not due to the fusion of the two β-α-β building blocks because an engineered "unfused" variant that breaks the covalent bond between the two units (to generate a heterohexamer) assumes the same asymmetric oligomerization state. It remains unknown how the different active site configurations contribute to the observed overall activities and whether the asymmetry has a biological purpose or role in the evolution of TSF members.

The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.

The Michael-type addition reaction is widely used in organic synthesis for carbon–carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon–carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon–carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence–function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary ‘Michaelases'. These ‘Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives., The Michael-type addition reaction is used for carbon-carbon bond formation; however biocatalytic methods for this reaction are rare. Here, the authors generate and exploit mutability landscapes of 4-oxalocrotonate tautomerase to direct the redesign of this promiscuous enzyme into enantio-complementary Michaelases.

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.

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.

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) ).

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.

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.

The vast majority of characterized oxygenases use bound cofactors to activate molecular oxygen to carry out oxidation chemistry. Here, we show that an enzyme of unknown activity, RhCC from Rhodococcus jostii RHA1, functions as an oxygenase, using 4-hydroxyphenylenolpyruvate as a substrate. This unique and complex reaction yields 3-hydroxy-3-(4-hydroxyphenyl)-pyruvate, 4-hydroxybenzaldehyde, and oxalic acid as major products. Incubations with H2(18)O, (18)O2, and a substrate analogue suggest that this enzymatic oxygenation reaction likely involves a peroxide anion intermediate. Analysis of sequence similarity and the crystal structure of RhCC (solved at 1.78 Å resolution) reveal that this enzyme belongs to the tautomerase superfamily. Members of this superfamily typically catalyze tautomerization, dehalogenation, or decarboxylation reactions rather than oxygenation reactions. The structure shows the absence of cofactors, establishing RhCC as a rare example of a redox-metal- and coenzyme-free oxygenase. This sets the stage to study the mechanistic details of cofactor-independent oxygen activation in the unusual context of the tautomerase superfamily.

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.

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.

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 beta-alpha-beta 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 H-1 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 2-013 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 pK(a) value that lies in between that of a typical tautomerase (pK(a) of Pro-I similar to 6) and that of cis-CaaD (pK(a) of Pro-I similar to 9). The shared activities and structural features, coupled with the intermediate pK(a) 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.

Macrophage migration inhibitory factor (M IF) is a multifunctional mammalian cytokine, which exhibits tautomerase and oxidoreductase activity. M IF homologues with pairwise sequence identities to human M IF ranging from 31% to 41% have been detected in various cyanobacteria. The gene encoding the M IF 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 phenylenol-pyruvate and (p-hydroxyphenyl)enolpyruvate at low levels. The N-terminal proline of PmMIF is critical for these reactions because the PIA 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 angstrom 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.

Graphical abstract An amino-terminal proline analogue: The enzyme 4-oxalocrotonate tautomerase (4-OT) is characterized by a catalytic N-terminal proline. The substitution of Pro-1 by a Gln, followed by in vivo removal of the initiating methionine by an engineered methionine aminopeptidase and cyclization of Gln-1 to form pyroglutamate, is a unique way to obtain a structural analogue of proline on the N-terminus of 4-OT., Highlights • The P1S variant of 4-OT was fully demethionylated by wild-type MetAP. • The P1H and P1Q variants of 4-OT were partially demethionylated by MetAP-∗TG. • Gln-1 is converted into pyro-Glu (pE) by spontaneous intramolecular cyclization. • Replacement of Pro-1 by Gln is a unique method to incorporate pE-1 in 4-OT., 4-Oxalocrotonate tautomerase (4-OT) catalyzes the enol-keto tautomerization of 2-hydroxymuconate, utilizing its N-terminal proline (Pro-1) as general base catalyst. Substituting Pro-1 with bulky or charged residues will result in poor or no post-translational removal of the translation-initiating methionine by the methionine aminopeptidase (MetAP) of the Escherichiacoli expression host. Here, we set out to investigate whether co-expression with previously engineered aminopeptidase MetAP-∗TG can be used to produce the P1S, P1H and P1Q variants of 4-OT in a demethionylated form. The P1S variant, which carries a small residue at the penultimate position (the first position after the initiating methionine), was found to be fully processed by wild-type MetAP. The P1S variant has low-level 2-hydroxymuconate tautomerase and promiscuous oxaloacetate decarboxylase activity. The P1Q and P1H variants of 4-OT, which carry bulky residues at the penultimate position, could only be obtained in a demethionylated form (a minor fraction of the purified protein is still composed of methionylated enzyme) by co-expression with MetAP-∗TG. Interestingly, the Gln-1 residue of the demethionylated P1Q variant undergoes intramolecular cyclization to form pyroglutamate (pE), yielding variant P1pE. Whereas the P1H/M1P2H mixture has low-level tautomerase activity, the P1pE/M1P2Q mixture has robust tautomerase activity. The substitution of Pro-1 by Gln, followed by removal of the initiating Met and cyclization of Gln-1 to form pE, is a unique way to obtain a structural analogue of proline on the N-terminus of 4-OT. This opens up new possibilities to study the importance of Pro-1 in recently discovered C–C bond-forming activities of this highly promiscuous tautomerase.