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Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

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.

Trichomonas vaginalis is the protist parasite that causes the most common, non-viral sexually transmitted infection called trichomonosis. Enolase is a moonlighting protein that apart from its canonical function as a glycolytic enzyme, serves as a plasminogen receptor on the cell surface of T. vaginalis and, in consequence, it has been stablished as a virulence factor in this parasite. In the Trichomonas vaginalis sequence database there are nine genes annotated as enolase. In this work, we analyzed these genes as well as their products. We found that seven out of nine genes might indeed perform enolase activity, whereas two genes might have been equivocally identified, or they might be pseudogenes. Furthermore, a combination of qRT-PCR and proteomic approaches was used to assess, for the first time, the expression of these genes in the highly virulent mexican isolate of T. vaginalis CNCD-147 at different iron concentrations. We could find peptides corresponding to enolases encoded by genes TVAG_464170, TVAG_043500 and TVAG_329460. Moreover, we identified two distinctive characteristics within enolases from Trichomonas vaginalis. One of them corresponds to three key substitutions within one of the loops of the active site, compared to host enolase. The other, is a unique N-terminal motif, composed of 15 to 18 residues, on all the potentially active enolases, whose function still has to be stablished. Both differential features merit further studies as potential drug and vaccine targets as well as diagnosis markers. These findings offer new possibilities to fight trichomonosis.

Proliferating cell nuclear antigen (PCNA) coordinates multi-enzymatic reactions by interacting with a variety of protein partners. Family I DNA ligases are multidomain proteins involved in sealing of DNA nicks during Okazaki fragment maturation and DNA repair. The interaction of DNA ligases with the inter-domain connector loop (IDCL) of PCNA through its PCNA interacting peptide (PIP box) is well studied but the role of the interacting surface between both proteins is not well characterized. In this work, we used a minimal DNA ligase I and two N-terminal deletions to establish that DNA binding and nick-sealing stimulation of DNA ligase I by PCNA are not solely dependent on the PIP box–IDCL interaction. We found that a truncated DNA ligase I with a deleted PIP box is stimulated by PCNA. Furthermore, the activity of a DNA ligase defective in DNA binding is rescued upon PCNA addition. Since the rate constants for single-turnover ligation for the full-length and truncated DNA ligases are not affected by PCNA, our data suggest that PCNA stimulation is achieved by increasing the affinity for nicked DNA substrate and not by increasing catalytic efficiency. Surprisingly C-terminal mutants of PCNA are not able to stimulate nick-sealing activity of Entamoeba histolytica DNA ligase I. Our data supports the notion that the C-terminal region of PCNA may be involved in promoting an allosteric transition in E. histolytica DNA ligase I from a spread-shaped to a ring-shaped structure. This study suggests that the ring-shaped PCNA is a binding platform able to stabilize co-evolved protein–protein interactions, in this case an interaction with DNA ligase I. This article is protected by copyright. All rights reserved.

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 A.

Background: Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3′ end of the primer. Methods Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p. Results The introduction of pathogenic variants C1188R (yV945R), and of a stop codon at residue T1199 (yT956X) abolished both polymerization and exonucleolysis in vitro. HsPolγ substitutions in residues D1184 (yD941), I1185 (yI942), K1191 (yK948) and D1196 (yD953) shifted the balance between polymerization and exonucleolysis in favor of exonucleolysis. HsPolγ pathogenic variants at residue K1191 (yK948) and D1184 (yD941) were capable of nucleotide incorporation albeit with reduced processivity. Structural analysis of mitochondrial DNAPs showed that residue HsPolγ-N864 is placed in an optimal distance to interact with the 3′ end of the primer and the phosphate backbone previous to the 3′ end. Amino acid changes in residue HsPolγ-N864 to Ala, Ser or Asp result in enzymes that did not decrease their polymerization activity on short templates but exhibited a substantial decrease for processive DNA synthesis. Conclusion Our data suggest that in mitochondrial DNA polymerases multiple amino acids are involved in the primer-stand stabilization.

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.

In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (β-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity.

Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5'-deoxyribose phosphate (5'-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5'-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5'-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5'-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer.

In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (β-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the redox environment in the chloroplast.

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.