4-oxalocrotonate Tautomerase Research Articles

Conformational Flexibility of the C-Terminal Region Influences Distal Active Site Residues Across the Tautomerase Superfamily

Consisting of more than 11,000 members distributed over five families, the tautomerase superfamily (TSF) is a large collection of proteins with diverse biological functions. While much attention has been given to individual TSF enzymes, a majority remain structurally and functionally uncharacterized. Given its large size, studying a representative member of each family offers a viable approach for extracting mechanistic insights applicable to the entire superfamily. In this study, cis-3-chloroacrylic acid dehalogenase (cis-CaaD), 5-carboxymethyl-2-hydroxymuconate isomerase (CHMI), malonate semialdehyde decarboxylase (MSAD), and 4-oxalocrotonate tautomerase (4-OT) were referenced against the well-studied macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) using triplicate 1 μs molecular dynamics (MD) simulations for a total of 18 μs. Through root mean square fluctuation (RMSF) measurements, correlation analyses, and comparisons to previous crystallographic structures, we reveal key mechanistic insights that promote the understanding of the catalytic activities in TSF. Collectively, our findings from these functionally diverse TSF proteins provide key information on allosteric coupling, long-range intra- and inter-subunit communications as well as structure–activity relationships that enable new studies in the superfamily.

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases.

Members of the 4-oxalocrotonate tautomerase (4-OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single β-α-β unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined β-α-β units and form trimers. A subset of sequences, double the length of the short 4-OTs, is found in the 4-OT subgroup. These "fused" 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4-OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the α- or β-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are αPro1 and βArg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180° relative to the other two heterodimers. The fusion of two subunits (α and β) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.

Biocatalytic Cascade Synthesis of Enantioenriched Epoxides and Triols from Biomass-Derived Synthons Driven by Specifically Designed Enzymes.

Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.

Formaldehyde reacts with N-terminal proline residues to give bicyclic aminals

Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.

Enantioselective conjugate addition of malonates to α,β-unsaturated aldehydes catalysed by 4-oxalocrotonate tautomerase.

The enantioselective conjugate addition of malonates to α,β-unsaturated aldehydes catalysed by 4-oxalocrotonate tautomerase is described. High conversions, high enantioselectivities, and good isolation yields were achieved for a range of substrates. We further completed a four-step synthesis of the antidepressant (+)-femoxetine by utilizing this reaction and an enzymatic reductive amination reaction.

Enantioselective Biocascade Catalysis with a Single Multifunctional Enzyme.

Asymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4-oxalocrotonate tautomerase (4-OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Enhancing the Peroxygenase Activity of a Cofactor-Independent Peroxyzyme by Directed Evolution Enabling Gram-Scale Epoxide Synthesis.

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H2 O2 with water being the sole by-product. These biocatalysts can provide 'green' routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4-oxalocrotonate tautomerase (4-OT), catalysing the epoxidation of a variety of α,β-unsaturated aldehydes with H2 O2 , is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4-OT. Herein we report the directed evolution of a tandem-fused 4-OT variant, which showed an ∼150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,β-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.

Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase.

The recent discovery of asymmetric arrangements of trimers in the tautomerase superfamily (TSF) adds structural diversity to this already mechanistically diverse superfamily. Classification of asymmetric trimers has previously been determined using X-ray crystallography. Here, native mass spectrometry (MS) and ultraviolet photodissociation (UVPD) are employed as an integrated strategy for more rapid and sensitive differentiation of symmetric and asymmetric trimers. Specifically, the unfolding of symmetric and asymmetric trimers initiated by collisional heating was probed using UVPD, which revealed unique gas-phase unfolding pathways. Variations in UVPD patterns from native-like, compact trimeric structures to unfolded, extended conformations indicate a rearrangement of higher-order structure in the asymmetric trimers that are believed to be stabilized by salt-bridge triads, which are absent from the symmetric trimers. Consequently, the symmetric trimers were found to be less stable in the gas phase, resulting in enhanced UVPD fragmentation overall and a notable difference in higher-order re-structuring based on the extent of hydrogen migration of protein fragments. The increased stability of the asymmetric trimers may justify their evolution and concomitant diversification of the TSF. Facilitating the classification of TSF members as symmetric or asymmetric trimers assists in delineating the evolutionary history of the TSF.

Structural insights into the substrate specificity of 5-chloro-2-hydroxymuconate tautomerase CnbG

The catechol meta-cleavage pathway is widely involved in the degradation of aromatic compounds, including those halogenated aromatic hydrocarbons and their derivatives. CnbG is a kind of 4-oxalocrotonate tautomerase (4-OT) located in the catechol meta-cleavage pathway, catalyzes the ketonization of cis,cis-5-chloro-2-hydroxymuconate and cis,cis-2-hydroxymuconate to yield 5-chloro-2-oxo-3-hexene-1,6-dioate and 2-oxo-3-hexene-1,6-dioate, and contributes to the degradation of 4-chloronitrobenzene and chlorobenzene in Comamonas testosteroni CNB-1. Yet, the reason why CnbG and those 4-OTs could recognize various substrates is not well explained. Here, we determined the crystal structure of CnbG at resolution of 2.0 Å and identified that the potential substrate pocket involved in four conserved residues, residues Pro1, Arg11, Arg39 and Trp50, but not five conserved residues as those reported in other 4-OTs. We also found the four conserved residues assemble different sequence patterns in different 4-OTs, indicating their potential roles in catalysis and substrate binding. Via molecular docking, we found the 5-chloro group was clamped by two residues and extended to the solvent, indicating a substrate binding mode that could bear the substitution of different groups in the 5-position. Our work extends the knowledge of the substrate specificity of enzymes in the catechol meta-cleavage pathway.

Comparative Metabolic Pathways Analysis and Subtractive Genomics Profiling to Prioritize Potential Drug Targets Against Streptococcus pneumoniae.

Streptococcus pneumoniae is a notorious pathogen that affects ∼450 million people worldwide and causes up to four million deaths per annum. Despite availability of antibiotics (i.e., penicillin, doxycycline, or clarithromycin) and conjugate vaccines (e.g., PCVs), it is still challenging to treat because of its drug resistance ability. The rise of antibiotic resistance in S. pneumoniae is a major source of concern across the world. Computational subtractive genomics is one of the most applied techniques in which the whole proteome of the bacterial pathogen is gradually reduced to a limited number of potential therapeutic targets. Whole-genome sequencing has greatly reduced the time required and provides more opportunities for drug target identification. The goal of this work is to evaluate and analyze metabolic pathways in serotype 14 of S. pneumonia to identify potential drug targets. In the present study, 47 potent drug targets were identified against S. pneumonia by employing the computational subtractive genomics approach. Among these, two proteins are prioritized (i.e., 4-oxalocrotonate tautomerase and Sensor histidine kinase uniquely present in S. pneumonia) as novel drug targets and selected for further structure-based studies. The identified proteins may provide a platform for the discovery of a lead drug candidate that may be capable of inhibiting these proteins and, therefore, could be helpful in minimizing the associated risk related to the drug-resistant S. pneumoniae. Finally, these enzymatic proteins could be of prime interest against S. pneumoniae to design rational targeted therapy.

Back Cover: Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C−C Bond‐Forming Enzyme (Angew. Chem. Int. Ed. 8/2022)

Gene fusion is an important natural process to create new enzymes from simpler ancestors. Gerrit J. Poelarends and co-workers have adopted this strategy in their Communication (e202113970) to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient enzyme for enantioselective Michael reactions. They designed a tandem fused 4-OT that has reduced structural symmetry, expanding its optimization potential by allowing independent sequence diversification of adjacent subunits, and thus enlarging the protein sequence space that can be travelled by directed evolution.

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme.

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4‐oxalocrotonate tautomerase (4‐OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem‐fused 4‐OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4‐OT was then subjected to eleven rounds of directed evolution to give variant 4‐OT(F11), which showed an up to 320‐fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4‐OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.

Combining Evolutionary Conservation and Quantum Topological Analyses To Determine Quantum Mechanics Subsystems for Biomolecular Quantum Mechanics/Molecular Mechanics Simulations.

Selection of residues and other molecular fragments for inclusion in the quantum mechanics (QM) region for QM/molecular mechanics (MM) simulations is an important step for these calculations. Here, we present an approach that combines protein sequence/structure evolution and electron localization function (ELF) analyses. The combination of these two analyses allows the determination of whether a residue needs to be included in the QM subsystem or can be represented by the MM environment. We have applied this approach on two systems previously investigated by QM/MM simulations, 4-oxalocrotonate tautomerase (4OT) and ten-eleven translocation-2 (TET2), that provide examples where fragments may or may not need to be included in the QM subsystem. Subsequently, we present the use of this approach to determine the appropriate QM subsystem to calculate the minimum energy path (MEP) for the reaction catalyzed by human DNA polymerase λ (Polλ) with a third cation in the active site. Our results suggest that the combination of protein evolutionary and ELF analyses provides insights into residue/molecular fragment selection for QM/MM simulations.

Evolutionary, genomic, and biogeographic characterization of two novel xenobiotics-degrading strains affiliated with Dechloromonas

Xenobiotics are generally known as man-made refractory organic pollutants widely distributed in various environments. For exploring the bioremediation possibility of xenobiotics, two novel xenobiotics-degrading strains affiliated with Azonexaceae were isolated. We report here the phylogenetics, genome, and geo-distribution of a novel and ubiquitous Azonexaceae species that primarily joins in the cometabolic process of some xenobiotics in natural communities. Strains s22 and t15 could be proposed as a novel species within Dechloromonas based on genomic and multi-phylogenetic analysis. Pan-genome analysis showed that the 63 core genes in Dechloromonas include genes for dozens of metabolisms such as nitrogen fixation protein (nifU), nitrogen regulatory protein (glnK), dCTP deaminase, C4-dicarboxylate transporter, and fructose-bisphosphate aldolase. Strains s22 and t15 have the ability to metabolize nitrogen, including nitrogen fixation, NirS-dependent denitrification, and dissimilatory nitrate reduction. Moreover, the novel species possesses the EnvZ-OmpR two-component system for controlling osmotic stress and QseC-QseB system for quorum sensing to rapidly sense environmental changes. It is intriguing that this new species has a series of genes for the biodegradation of some xenobiotics such as azathioprine, 6-Mercaptopurine, trinitrotoluene, chloroalkane, and chloroalkene. Specifically, glutathione S-transferase (GST) and 4-oxalocrotonate tautomerase (praC) in this novel species play important roles in the detoxification metabolism of some xenobiotics like dioxin, trichloroethene, chloroacetyl chloride, benzo[a]pyrene, and aflatoxin B1. Using data from GenBank, DDBJ and EMBL databases, we also demonstrated that members of this novel species were found globally in plants (e.g. rice), guts (e.g. insect), pristine and contaminated regions. Given these data, Dechloromonas sp. strains s22 and t15 take part in the biodegradation of some xenobiotics through key enzymes.

A guide to benchmarking enzymatically catalysed reactions: the importance of accurate reference energies and the chemical environment

We explore two significant factors on the outcomes of benchmark studies for enzymatically catalyzed reactions, namely the level of theory of the benchmarks and the size of the model system used to represent the enzyme active site. For the benchmarks, we compare two potential alternatives to canonical coupled cluster results for situations where CCSD(T) is computationally too demanding: a strategy to estimate finite basis set coupled cluster values and the local-correlation DLPNO-CCSD(T) method at the complete basis set limit. We confirm the high accuracy of DLPNO-CCSD(T) used with tight thresholds. We also show that notable differences can be seen when using both sets of references for a benchmark study, with absolute deviations from the higher-quality references generally smaller than those from lower-quality ones as well as changes in the ranking of the assessed methods. For geometries, we test three models for the active site of 4-oxalocrotonate tautomerase: one typical of the QM region that may be used in QM/MM studies, and two smaller variants that neglect the surrounding chemical environment. Benchmarking of 12 density functionals known to perform well on enzymatically catalyzed reactions shows inconsistent performance of each method across the three models, contradicting the common idea that small representative systems can be used to accurately assess the applicability of low-level methods for larger biochemical applications. Our findings shall serve as a reminder on the standards that should be adhered to in benchmark studies, and as a guide for future studies, both on enzyme-related and other chemical problems.

Kinetic and Structural Analysis of Two Linkers in the Tautomerase Superfamily: Analysis and Implications

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.

Spatio-temporal resolution of taxonomic and functional microbiome of Lonar soda lake of India reveals metabolic potential for bioremediation

Lonar Lake, India; a hypersaline and hyperalkaline extremophilic ecosystem having a unique microbial population has been rarely explored for bioremediation aspects. MinION-based shotgun sequencing was used to comprehensively compare the microbial diversity and functional potential of xenobiotic degradation pathways with seasonal changes. Proteobacteria and Firmicutes were prevalent bacterial phyla in the pre-monsoon and post-monsoon samples. Functional analysis from SEED-subsystem and KEGG database revealed 28 subsystems and 18 metabolic pathways for the metabolism of aromatic compounds and xenobiotic biodegradation respectively. Occurrence of N-phenyl alkanoic, benzoate, biphenyl, chloroaromatic, naphthalene, and phenol degradation genes depicted varied abundance in the pre-monsoon and post-monsoon samples. Further, KEGG analysis indicated nitrotoluene degradation pathway (ko00633) abundant in post-monsoon samples, and the benzoate degradation pathway (ko00362) predominant in 19LN4S (pre-monsoon) than 18LN7S (post-monsoon) samples. The abundant genes for benzoate degradation were pcaI: 3-oxoadipate CoA-transferase, alpha subunit, pcaH: protocatechuate 3,4-dioxygenase, beta subunit, and pcaB: 3-carboxy-cis, cis-muconate cycloisomerase, and 4-oxalocrotonate tautomerase. This metagenomic study provides a unique blueprint of hitherto unexplored xenobiotic biodegradation genes/pathways in terms of seasonal variations in the Lonar Lake, and warrants active exploitation of microbes for bioremediation purposes.

Structural Basis for the Asymmetry of a 4-Oxalocrotonate Tautomerase Trimer.

Tautomerase superfamily (TSF) members are constructed from a single β-α-β unit or two consecutively joined β-α-β units. This pattern prevails throughout the superfamily consisting of more than 11000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4-OT) subgroup and trimers are found in the other four subgroups. One exception is a subset of sequences that are double the length of the short 4-OTs in the 4-OT subgroup, where the coded proteins form trimers. Characterization of two members revealed an interesting dichotomy. One is a symmetric trimer, whereas the other is an asymmetric trimer. One monomer is flipped 180° relative to the other two monomers so that three unique protein-protein interfaces are created that are composed of different residues. A bioinformatics analysis of the fused 4-OT subset shows a further division into two clusters with a total of 133 sequences. The analysis showed that members of one cluster (86 sequences) have more salt bridges if the asymmetric trimer forms, whereas the members of the other cluster (47 sequences) have more salt bridges if the symmetric trimer forms. This hypothesis was examined by the kinetic and structural characterization of two proteins within each cluster. As predicted, all four proteins function as 4-OTs, where two assemble into asymmetric trimers (designated R7 and F6) and two form symmetric trimers (designated W0 and Q0). These findings can be extended to the other sequences in the two clusters in the fused 4-OT subset, thereby annotating their oligomer properties and activities.

In Situ Acetaldehyde Synthesis for Carboligation Reactions.

The enzyme 4‐oxalocrotonate tautomerase (4‐OT) can promiscuously catalyze various carboligation reactions using acetaldehyde as a nucleophile. However, the highly reactive nature of acetaldehyde requires intricate handling, which can impede its usage in practical synthesis. Therefore, we investigated three enzymatic routes to synthesize acetaldehyde in situ in one‐pot cascade reactions with 4‐OT. Two routes afforded practical acetaldehyde concentrations, using an environmental pollutant, trans‐3‐chloroacrylic acid, or a bio‐renewable, ethanol, as starting substrate. These routes can be combined with 4‐OT catalyzed Michael‐type additions and aldol condensations in one pot. This modular systems biocatalysis methodology provides a stepping stone towards the development of larger artificial metabolic networks for the practical synthesis of important chemical synthons.

Selective Colorimetric "Turn-On" Probe for Efficient Engineering of Iminium Biocatalysis.

The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric “turn-on” probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level “Michaelase” activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis.