Triosephosphate Isomerase: The Crippling Effect of the P168A/I172A Substitution at the Heart of an Enzyme Active Site.

The P168 and I172 side chains sit at the heart of the active site of triosephosphate isomerase (TIM) and play important roles in the catalysis of the isomerization reaction. The phosphodianion of substrate glyceraldehyde 3-phosphate (GAP) drives a conformational change at the TIM that creates a steric interaction with the P168 side chain that is relieved by the movement of P168 that carries the basic E167 side chain into a clamp that consists of the hydrophobic I172 and L232 side chains. The P168A/I172A substitution at TIM from Trypanosoma brucei brucei (TbbTIM) causes a large 120,000-fold decrease in kcat for isomerization of GAP that eliminates most of the difference in the reactivity of TIM compared to the small amine base quinuclidinone for deprotonation of catalyst-bound GAP. The I172A substitution causes a > 2-unit decrease in the pKa of the E167 carboxylic acid in a complex to the intermediate analog PGA, but the P168A substitution at the I172A variant has no further effect on this pKa. The P168A/I172A substitutions cause a 5-fold decrease in Km for the isomerization of GAP from a 0.9 kcal/mol stabilization of the substrate Michaelis complexes. The results show that the P168 and I172 side chains play a dual role in destabilizing the ground-state Michaelis complex to GAP and in promoting stabilization of the transition state for substrate isomerization. This is consistent with an important role for these side chains in an induced fit reaction mechanism [Richard, J. P. (2022) Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution. Biochemistry 61, 1533-1542].

Repurposing of rabeprazole as an anti-Trypanosoma cruzi drug that targets cellular triosephosphate isomerase.

Trypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi, but cellular enzyme inhibition has yet to be established. This study demonstrates that rabeprazole inhibits both cell viability and triosephosphate isomerase activity in T. cruzi epimastigotes. Our results show that rabeprazole has an IC50 of 0.4 µM, which is 14.5 times more effective than benznidazole. Additionally, we observed increased levels of methyl-glyoxal and advanced glycation end products after the inhibition of cellular triosephosphate isomerase by rabeprazole. Finally, we demonstrate that the inactivation mechanisms of rabeprazole on triosephosphate isomerase of T. cruzi can be achieved through the derivatization of three of its four cysteine residues. These results indicate that rabeprazole is a promising candidate against American trypanosomiasis.

The Role of Asn11 in Catalysis by Triosephosphate Isomerase.

Four catalytic amino acids at triosephosphate isomerase (TIM) are highly conserved: N11, K13, H95, and E167. Asparagine 11 is the last of these to be characterized in mutagenesis studies. The ND2 side chain atom of N11 is hydrogen bonded to the O-1 hydroxyl of enzyme-bound dihydroxyacetone phosphate (DHAP), and it sits in an extended chain of hydrogen-bonded side chains that includes T75' from the second subunit. The N11A variants of wild-type TIM from Trypanosoma brucei brucei (TbbTIM) and Leishmania mexicana (LmTIM) undergo dissociation from the dimer to monomer under our assay conditions. Values of Kas = 8 × 103 and 1 × 106 M-1, respectively, were determined for the conversion of monomeric N11A TbbTIM and LmTIM into their homodimers. The N11A substitution at the variant of LmTIM previously stabilized by the E65Q substitution gives the N11A/E65Q variant that is stable to dissociation under our assay conditions. The X-ray crystal structure of N11A/E65Q LmTIM shows an active site that is essentially superimposable on that for wild-type TbbTIM, which also has a glutamine at position 65. A comparison of the kinetic parameters for E65Q LmTIM and N11A/E65Q LmTIM-catalyzed reactions of (R)-glyceraldehyde 3-phosphate (GAP) and (DHAP) shows that the N11A substitution results in a (13-14)-fold decrease in kcat/Km for substrate isomerization and a similar decrease in kcat for DHAP but only a 2-fold decrease in kcat for GAP.

Kinetics and mechanism for enzyme-catalyzed reactions of substrate pieces.

The most important difference between enzyme and small molecule catalysts is that only enzymes utilize the large intrinsic binding energies of nonreacting portions of the substrate in stabilization of the transition state for the catalyzed reaction. A general protocol is described to determine the intrinsic phosphodianion binding energy for enzymatic catalysis of reactions of phosphate monoester substrates, and the intrinsic phosphite dianion binding energy in activation of enzymes for catalysis of phosphodianion truncated substrates, from the kinetic parameters for enzyme-catalyzed reactions of whole and truncated substrates. The enzyme-catalyzed reactions so-far documented that utilize dianion binding interactions for enzyme activation; and, their phosphodianion truncated substrates are summarized. A model for the utilization of dianion binding interactions for enzyme activation is described. The methods for the determination of the kinetic parameters for enzyme-catalyzed reactions of whole and truncated substrates, from initial velocity data, are described and illustrated by graphical plots of kinetic data. The results of studies on the effect of site-directed amino acid substitutions at orotidine 5'-monophosphate decarboxylase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase provide strong support for the proposal that these enzymes utilize binding interactions with the substrate phosphodianion to hold the protein catalysts in reactive closed conformations.

Energetically unfavorable protein angles: Exploration of a conserved dihedral angle in triosephosphate isomerase.

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ = +51/Ψ = -143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted ß-sheet dihedral values of Φ = -135/Ψ = +170 but experiments show a 1000-fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation.

Triosephosphate isomerase (TPI) performs the 5th step in glycolysis, operates near the limit of diffusion, and is involved in "moonlighting" functions. Its dimer was found singly phosphorylated at Ser20 (pSer20) in human cells, with this post-translational modification (PTM) showing context-dependent stoichiometry and loss under oxidative stress. We generated synthetic pSer20 proteoforms using cell-free protein synthesis that showed enhanced TPI activity by 4-fold relative to unmodified TPI. Molecular dynamics simulations show that the phosphorylation enables a channel to form that shuttles substrate into the active site. Refolding, kinetic, and crystallographic analyses of point mutants including S20E/G/Q indicate that hetero-dimerization and subunit asymmetry are key features of TPI. Moreover, characterization of an endogenous human TPI tetramer also implicates tetramerization in enzymatic regulation. S20 is highly conserved across eukaryotic TPI, yet most prokaryotes contain E/D at this site, suggesting that phosphorylation of human TPI evolved a new switch to optionally boost an already fast enzyme. Overall, complete characterization of TPI shows how endogenous proteoform discovery can prioritize functional versus bystander PTMs.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution.

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein-substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

Highly Similar Sequence and Structure Yet Different Biophysical Behavior: A Computational Study of Two Triosephosphate Isomerases.

Homodimeric triosephosphate isomerases (TIMs) from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar amino-acid sequences and three-dimensional structures. However, several of their biophysical parameters, such as their susceptibility to sulfhydryl agents and their reactivation speed after being denatured, have significant differences. The causes of these differences were explored with microsecond-scale molecular dynamics (MD) simulations of three different TIM proteins: TcTIM, TbTIM, and a chimeric protein, Mut1. We examined their electrostatic interactions and explored the impact of simulation length on them. The same salt bridge between catalytic residues Lys 14 and Glu 98 was observed in all three proteins, but key differences were found in other interactions that the catalytic amino acids form. In particular, a cation-π interaction between catalytic amino acids Lys 14 and His 96 and both a salt bridge and a hydrogen bond between catalytic Glu 168 and residue Arg 100 were only observed in TcTIM. Furthermore, although TcTIM forms less hydrogen bonds than TbTIM and Mut1, its hydrogen bond network spans almost the entire protein, connecting the residues in both monomers. This work provides new insight into the mechanisms that give rise to the different behavior of these proteins. The results also show the importance of long simulations.

Recent Advances in the Development of Triose Phosphate Isomerase Inhibitors as Antiprotozoal Agents.

BACKGROUND: Parasitic diseases caused by protozoa, such as Chagas disease, leishmaniasis, malaria, African trypanosomiasis, amoebiasis, trichomoniasis, and giardiasis, are considered serious public health problems in developing countries. Drug resistance among parasites justifies the search for new therapeutic drugs, and the identification of new targets becomes a valuable approach. In this scenario, the glycolysis pathway, which converts glucose into pyruvate, plays an important role in the protozoa energy supply, and it is therefore considered a promising target. In this pathway, triose phosphate isomerase (TIM) plays an essential role in efficient energy production. Furthermore, protozoa TIM shows structural differences with human enzyme counterparts, suggesting the possibility of obtaining selective inhibitors. Therefore, TIM is considered a valid approach to develop new antiprotozoal agents, inhibiting the glycolysis in the parasite. OBJECTIVE: In this review, we discuss the drug design strategies, structure-activity relationship, and binding modes of outstanding TIM inhibitors against Trypanosoma cruzi, Trypanosoma brucei, Plasmodium falciparum, Giardia lamblia, Leishmania mexicana, Trichomonas vaginalis, and Entamoeba histolytica. RESULTS: TIM inhibitors have mainly shown aromatic systems and symmetrical structure, where the size and type of heteroatom are important for enzyme inhibition. This inhibition is mainly based on the interaction with i) the interfacial region of TIM inducing changes on the quaternary and tertiary structure or ii) with the TIM catalytic region, the main pathways that disable the catalytic activity of the enzyme. CONCLUSION: Benzothiazole, benzoxazole, benzimidazole, and sulfhydryl derivatives stand out as TIM inhibitors. In silico and in vitro studies have demonstrated that the inhibitors bind mainly at the TIM dimer interface. In this review, the development of new TIM inhibitors as antiprotozoal drugs is demonstrated as an important pharmaceutical strategy that may lead to new therapies for these ancient parasitic diseases.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency.

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

Linear Free Energy Relationships for Enzymatic Reactions: Fresh Insight from a Venerable Probe.

Linear free energy relationships (LFERs) for substituent effects on reactions that proceed through similar transition states provide insight into transition state structures. A classical approach to the analysis of LFERs showed that differences in the slopes of Brønsted correlations for addition of substituted alkyl alcohols to ring-substituted 1-phenylethyl carbocations and to the ß-galactopyranosyl carbocation intermediate of reactions catalyzed by ß-galactosidase provide evidence that the enzyme catalyst modifies the curvature of the energy surface at the saddle point for the transition state for nucleophile addition. We have worked to generalize the use of LFERs in the determination of enzyme mechanisms. The defining property of enzyme catalysts is their specificity for binding the transition state with a much higher affinity than the substrate. Triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase (OMPDC), and glycerol 3-phosphate dehydrogenase (GPDH) show effective catalysis of reactions of phosphorylated substrates and strong phosphite dianion activation of reactions of phosphodianion truncated substrates, with rate constants kcat/Km (M-1 s-1) and kcat/KdKHPi (M-2 s-1), respectively. Good linear logarithmic correlations, with a slope of 1.1, between these kinetic parameters determined for reactions catalyzed by five or more variant forms of each catalyst are observed, where the protein substitutions are mainly at side chains which function to stabilize the cage complex between the enzyme and substrate. This shows that the enzyme-catalyzed reactions of a whole substrate and substrate pieces proceed through transition states of similar structures. It provides support for the proposal that the dianion binding energy of whole phosphodianion substrates and of phosphite dianion is used to drive the conversion of these protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes that show the same activity toward catalysis of the reactions of whole and phosphodianion truncated substrates. There is a good linear correlation, with a slope of 0.73, between values of the dissociation constants log Ki for release of the transition state analog phosphoglycolate (PGA) trianion and log kcat/Km for isomerization of GAP for wild-type and variants of TIM. This correlation shows that the substituted amino acid side chains act to stabilize the complex between TIM and the PGA trianion and that ca. 70% of this stabilization is observed at the transition state for substrate deprotonation. The correlation provides evidence that these side chains function to enhance the basicity of the E165 side chain of TIM, which deprotonates the bound carbon acid substrate. There is a good linear correlation, with a slope of 0.74, between the values of ΔG‡ and ΔG° determined by electron valence bond (EVB) calculations to model deprotonation of dihydroxyacetone phosphate (DHAP) in water and when bound to wild-type and variant forms of TIM to form the enediolate reaction intermediate. This correlation provides evidence that the stabilizing interactions of the transition state for TIM-catalyzed deprotonation of DHAP are optimized by placement of amino acid side chains in positions that provide for the maximum stabilization of the charged reaction intermediate, relative to the neutral substrate.

Enhanced Antigiardial Effect of Omeprazole Analog Benzimidazole Compounds.

Giardiasis is a diarrheal disease that is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy are common, and have adverse effects and increased resistance of the parasite to the drug, generating the need to find new alternative treatments. In this study, we synthesized a series of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were confirmed through mass, 1H NMR, and 13C NMR techniques. The in vitro efficacy compounds against Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for 2 h at 37 °C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target compounds, H-BZM2, O2N-BZM7, and O2N-BZM9 had greater antigiardial activity (IC50: 36, 14, and 17 µM on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2, and 2.8 M-1 s-1) respectively, loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein. Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target.

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.

Triosephosphate isomerase deficiency: Effect of F240L mutation on enzyme structure.

Eleven missense mutations have been describe in human triosephosphate isomerase (TPI), affecting its catalytic function. Several of these mutations generate triosephosphate isomerase deficiency, the consequences of which can in some cases be lethal. The missense F240L mutation was found in a Hungarian patient showing symptoms of chronic hemolytic anemia and neuromuscular dysfunction. In vitro studies using a recombinant version of this mutant showed that it affects kinetic parameters, thermal stability and dimeric stability. Using X-ray crystal structures, the present paper describes how this mutation affected the flexibility of catalytic residues K13 and part of the (ß/α) 8-barrel fold facing the dimeric interface in the TPI.

Crystallographic Studies of Triosephosphate Isomerase from Schistosoma mansoni.

Protein structure determination by X-ray crystallography guides structure-function and rational drug design studies. Helminths cause devastating diseases, including schistosomiasis that affects over one-third of the human population. Trematodes from the genus Schistosoma heavily depend on glycolysis; thus enzymes involved in this metabolic pathway are potential drug targets. Here we present a protocol to obtain crystal structures of recombinantly expressed triosephosphate isomerase from S. mansoni (SmTPI) that diffracted in house to a resolution of 2 Å.

Native aggregation is a common feature among triosephosphate isomerases of different species.

Triosephosphate isomerase (TIM) is an enzyme of the glycolysis pathway which exists in almost all types of cells. Its structure is the prototype of a motif called TIM-barrel or (α/ß)8 barrel, which is the most common fold of all known enzyme structures. The simplest form in which TIM is catalytically active is a homodimer, in many species of bacteria and eukaryotes, or a homotetramer in some archaea. Here we show that the purified homodimeric TIMs from nine different species of eukaryotes and one of an extremophile bacterium spontaneously form higher order aggregates that can range from 3 to 21 dimers per macromolecular complex. We analysed these aggregates with clear native electrophoresis with normal and inverse polarity, blue native polyacrylamide gel electrophoresis, liquid chromatography, dynamic light scattering, thermal shift assay and transmission electron and fluorescence microscopies, we also performed bioinformatic analysis of the sequences of all enzymes to identify and predict regions that are prone to aggregation. Additionally, the capacity of TIM from Trypanosoma brucei to form fibrillar aggregates was characterized. Our results indicate that all the TIMs we studied are capable of forming oligomers of different sizes. This is significant because aggregation of TIM may be important in some of its non-catalytic moonlighting functions, like being a potent food allergen, or in its role associated with Alzheimer's disease.

Crystal structures of Triosephosphate Isomerases from Taenia solium and Schistosoma mansoni provide insights for vaccine rationale and drug design against helminth parasites.

Triosephosphate isomerases (TPIs) from Taenia solium (TsTPI) and Schistosoma mansoni (SmTPI) are potential vaccine and drug targets against cysticercosis and schistosomiasis, respectively. This is due to the dependence of parasitic helminths on glycolysis and because those proteins elicit an immune response, presumably due to their surface localization. Here we report the crystal structures of TsTPI and SmTPI in complex with 2-phosphoglyceric acid (2-PGA). Both TPIs fold into a dimeric (ß-α)8 barrel in which the dimer interface consists of α-helices 2, 3, and 4, and swapping of loop 3. TPIs from parasitic helminths harbor a region of three amino acids knows as the SXD/E insert (S155 to E157 and S157 to D159 in TsTPI and SmTPI, respectively). This insert is located between α5 and ß6 and is proposed to be the main TPI epitope. This region is part of a solvent-exposed 310-helix that folds into a hook-like structure. The crystal structures of TsTPI and SmTPI predicted conformational epitopes that could be used for vaccine design. Surprisingly, the epitopes corresponding to the SXD/E inserts are not the ones with the greatest immunological potential. SmTPI, but not TsTPI, habors a sole solvent exposed cysteine (SmTPI-S230) and alterations in this residue decrease catalysis. The latter suggests that thiol-conjugating agents could be used to target SmTPI. In sum, the crystal structures of SmTPI and TsTPI are a blueprint for targeted schistosomiasis and cysticercosis drug and vaccine development.

Phylogenetic spread of sequence data affects fitness of consensus enzymes: Insights from triosephosphate isomerase.

The concept of consensus in multiple sequence alignments (MSAs) has been used to design and engineer proteins previously with some success. However, consensus design implicitly assumes that all amino acid positions function independently, whereas in reality, the amino acids in a protein interact with each other and work cooperatively to produce the optimum structure required for its function. Correlation analysis is a tool that can capture the effect of such interactions. In a previously published study, we made consensus variants of the triosephosphate isomerase (TIM) protein using MSAs that included sequences form both prokaryotic and eukaryotic organisms. These variants were not completely native-like and were also surprisingly different from each other in terms of oligomeric state, structural dynamics, and activity. Extensive correlation analysis of the TIM database has revealed some clues about factors leading to the unusual behavior of the previously constructed consensus proteins. Among other things, we have found that the more ill-behaved consensus mutant had more broken correlations than the better-behaved consensus variant. Moreover, we report three correlation and phylogeny-based consensus variants of TIM. These variants were more native-like than the previous consensus mutants and considerably more stable than a wild-type TIM from a mesophilic organism. This study highlights the importance of choosing the appropriate diversity of MSA for consensus analysis and provides information that can be used to engineer stable enzymes.

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.