Oxidation Of Dithiols Research Articles

Computational approaches for molecular characterization and structure-based functional elucidation of a hypothetical protein from Mycobacterium tuberculosis.

Adaptation of infections and hosts has resulted in several metabolic mechanisms adopted by intracellular pathogens to combat the defense responses and the lack of fuel during infection. Human tuberculosis caused by Mycobacterium tuberculosis (MTB) is the world's first cause of mortality tied to a single disease. This study aims to characterize and anticipate potential antigen characteristics for promising vaccine candidates for the hypothetical protein of MTB through computational strategies. The protein is associated with the catalyzation of dithiol oxidation and/or disulfide reduction because of the protein's anticipated disulfide oxidoreductase properties. This investigation analyzed the protein's physicochemical characteristics, protein-protein interactions, subcellular locations, anticipated active sites, secondary and tertiary structures, allergenicity, antigenicity, and toxicity properties. The protein has significant active amino acid residues with no allergenicity, elevated antigenicity, and no toxicity.

L-Methionine Selenoxide as an Oxidizing and Deprotection Reagent for the Synthesis of Multiple Disulfide Bonds in Peptides.

The regioselective synthesis of multiple disulfide bonds in peptides has been a significant challenge in synthetic peptide chemistry. In this work, two disulfide bonds in peptides were regioselectively synthesized via an approach of MetSeO oxidation and deprotection reaction (SeODR), in which the first disulfide bond was constructed through oxidation of dithiol by MetSeO in a neutral buffer, and the second disulfide bond was then directly constructed through the deprotection of two Acm groups or one Acm group and one Thz group by MetSeO in acidic media. Synthesis of two disulfide bonds by the SeODR approach was achieved through a one-pot manner. Moreover, the SeODR approach is compatible with the synthesis of peptides containing methionine residues. Both H+ and Br- drastically promoted the reaction rate of SeODR. The mechanistic picture for the SeODR approach was delineated, in which the formation of a stable Se-X-S bridge as the transition state plays a critical role. The SeODR approach was also utilized to construct the three disulfide bonds in linaclotide, conferring a reasonable yield.

A novel soybean protein disulphide isomerase family protein possesses dithiol oxidation activity: identification and characterization of GmPDIL6.

Secretory and membrane proteins synthesized in the endoplasmic reticulum (ER) are folded with intramolecular disulphide bonds, viz. oxidative folding, catalysed by the protein disulphide isomerase (PDI) family proteins. Here, we identified a novel soybean PDI family protein, GmPDIL6. GmPDIL6 has a single thioredoxin-domain with a putative N-terminal signal peptide and an active centre (CKHC). Recombinant GmPDIL6 forms various oligomers binding iron. Oligomers with or without iron binding and monomers exhibited a dithiol oxidase activity level comparable to those of other soybean PDI family proteins. However, they displayed no disulphide reductase and extremely low oxidative refolding activity. Interestingly, GmPDIL6 was mainly expressed in the cotyledon during synthesis of seed storage proteins and GmPDIL6 mRNA was up-regulated under ER stress. GmPDIL6 may play a role in the formation of disulphide bonds in nascent proteins for oxidative folding in the ER.

Nitric oxide regulates chlorophyllide biosynthesis and singlet oxygen generation differently between Arabidopsis and barley

Nitric oxide (NO) has a general inhibitory effects on chlorophyll biosynthesis, especially to the step of 5-aminolevulinic acid (ALA) biosynthesis and protochlorophyllide (Pchlide) to chlorophyllide (Chlide) conversion (responsible by the NADPH:Pchlide oxidoreductase POR). Previous study suggested that barley large POR aggregates may be generated by dithiol oxidation of cysteines of two POR monomers, which can be disconnected by some reducing agents. POR aggregate assembly may be correlated with seedling greening in barley, but not in Arabidopsis. Thus, NO may affect POR activity and seedling greening differently between Arabidopsis and barley. We proved this assumption by non-denaturing gel-analysis and reactive oxygen species (ROS) monitoring during the greening. NO treatments cause S-nitrosylation to POR cysteine residues and disassembly of POR aggregates. This modification reduces POR activity and induces Pchlide accumulation and singlet oxygen generation upon dark-to-high-light shift (and therefore inducing photobleaching lesions) in barley leaf apex, but not in Arabidopsis seedlings. ROS staining and ROS-related-gene expression detection confirmed that superoxide anion and singlet oxygen accumulated in barley etiolated seedlings after the NO treatments, when exposed to a fluctuating light. The data suggest that POR aggregate assembly may be correlated with barley chlorophyll biosynthesis and redox homeostasis during greening. Cysteine S-nitrosylation may be one of the key reasons for the NO-induced inhibition to chlorophyll biosynthetic enzymes.

Glutaredoxins employ parallel monothiol-dithiol mechanisms to catalyze thiol-disulfide exchanges with protein disulfides.

Glutaredoxins (Grxs) are a family of glutathione (GSH)-dependent thiol-disulfide oxidoreductases. They feature GSH-binding sites that directly connect the reversible redox chemistry of protein thiols to the abundant cellular nonprotein thiol pool GSSG/GSH. This work studied the pathways for oxidation of protein dithiols P(SH)2 and reduction of protein disulfides P(SS) catalyzed by Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The metal-binding domain HMA4n(SH)2 was chosen as substrate as it contains a solvent-exposed CysCys motif. Quenching of the reactions with excess iodoacetamide followed by protein speciation analysis via ESI-MS allowed interception and characterization of both substrate and enzyme intermediates. The enzymes shuttle between three catalytically-competent forms (Grx(SH)(S-), Grx(SH)(SSG) and Grx(SS)) and employ conserved parallel monothiol and dithiol mechanisms. Experiments with dithiol and monothiol versions of both Grx enzymes demonstrate which monothiol (plus GSSG or GSH) or dithiol pathways dominate a specific oxidation or reduction reaction. Grxs are shown to be a class of versatile enzymes with diverse catalytic functions that are driven by specific interactions with GSSG/GSH.

Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Adsorption and thermal stability of 1,4 benzenedimethanethiol on InP(110)

Self-assembly of dithiol molecules is of interest because these can be used as linkers between metallic or semiconductor entities and thus employed in molecular electronics and plasmonic applications, or for building complex heterostructures. Here we focus on dithiol self-assembly by evaporation in vacuum, a method that could circumvent the dithiol oxidation that can occur in solution. We present a high resolution X-ray photoelectron spectroscopy (XPS) and an ion scattering study of adsorption and desorption of 1,4-benzenedimethanethiol on InP(110) as a function of exposure and sample temperature. Results for InP are compared to those on Au(111) and found to differ due to formation of a thick BDMT layer at room temperature, resulting from extra molecules sticking on top of the self-assembled monolayer. This may play an adverse effect in some afore-mentioned applications as in molecular electronics. We furthermore study the evolution of the dithiol film with sample temperature and the elements remaining at the surface after annealing and delineate initial coverage dependent effects.

Selective and ratiometric fluorescent trapping and quantification of protein vicinal dithiols and in situ dynamic tracing in living cells.

Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.

Synthesis and Application of an Environmentally Insensitive Cy3-Based Arsenical Fluorescent Probe To Identify Adaptive Microbial Responses Involving Proximal Dithiol Oxidation

Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable in vivo measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable thiol-reactive affinity probe (TRAP) consisting of a monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP_Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen and light levels in the photosynthetic microbe Synechococcus sp. PCC7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative protein modifications involving methionine sulfoxide and nitrotyrosine. These results suggest that the redox status of proximal cysteines responds to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage.

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.

H5[PMo10V2O40] as a Green, Reusable, and Highly Efficient Catalyst for the Oxidation of Dithiols in Intermolecular Reactions Using Permanganate as an Oxidizing Reagent

[H5PV2Mo10O40] catalyzed oxidation of dithiols to related cyclic disulfides. Potassium permanganate as an oxidizing reagent under mild conditions was carried out in this reaction. This system provides an efficient, convenient, and practical method for the syntheses of cyclic disulfides. In this work, the comparisons among the Keggin and Dawson ions are addressed in term of relative stability hardness and acidity.

Mitochondrial Dysfunction Induced by Different Organochalchogens Is Mediated by Thiol Oxidation and Is Not Dependent of the Classical Mitochondrial Permeability Transition Pore Opening

Ebselen (Ebs) and diphenyl diselenide [(PhSe)(2)] readily oxidize thiol groups. Here we studied mitochondrial swelling changes in mitochondrial potential (Deltapsim), NAD(P)H oxidation, reactive oxygen species production, protein aggregate formation, and oxygen consumption as ending points of their in vitro toxicity. Specifically, we tested the hypothesis that organochalchogens toxicity could be associated with mitochondrial dysfunction via oxidation of vicinal thiol groups that are known to be involved in the regulation of mitochondrial permeability (Petronilli et al. J. Biol. Chem., 269; 16638; 1994). Furthermore, we investigated the possible mechanism(s) by which these organochalchogens could disrupt liver mitochondrial function. Ebs and (PhSe)(2) caused mitochondrial depolarization and swelling in a concentration-dependent manner. Furthermore, both organochalchogens caused rapid oxidation of the mitochondrial pyridine nucleotides (NAD(P)H) pool, likely reflecting the consequence and not the cause of increased mitochondrial permeability (Costantini, P., Chernyak, B. V., Petronilli, V., and Bernardi, P. (1996). Modulation of the mitochondrial permeability transition pore (PTP) by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271, 6746-6751). The organochalchogens-induced mitochondrial dysfunction was prevented by the reducing agent dithiothreitol (DTT). Ebs- and (PhSe)(2)-induced mitochondrial depolarization and swelling were unchanged by ruthenium red (4microM), butylated hydroxytoluene (2.5microM), or cyclosporine A (1microM). N-ethylmaleimide enhanced the organochalchogens-induced mitochondrial depolarization, without affecting the magnitude of the swelling response. In contrast, iodoacetic acid did not modify the effects of Ebs or (PhSe)(2) on the mitochondria. Additionally, Ebs and (PhSe)(2) decreased the basal 2' 7' dichlorofluorescin diacetate (H(2)-DCFDA) oxidation and oxygen consumption rate in state 3 and increased it during the state 4 of oxidative phosphorylation and induced the formation of protein aggregates, which were prevented by DTT. However, DTT failed to reverse the formation of protein aggregates, when it was added after a preincubation of liver mitochondria with Ebs or (PhSe)(2). Similarly, DTT did not reverse the Ebs- or (PhSe)(2)-induced Deltapsim collapse or swelling, when it was added after a preincubation period of mitochondria with chalcogenides. These results show that Ebs and (PhSe)(2) can effectively induce mitochondrial dysfunction and suggest that effects of these compounds are associated with mitochondrial thiol groups oxidation. The inability of cyclosporine A to reverse the Ebs- and (PhSe)(2)-induced mitochondrial effects suggests that the redox-regulated mitochondrial permeability transition (MPT) pore was mechanistically regulated in a manner that is distinct from the classical MPT pore.

Optical Monitoring of Gases with Cholesteric Liquid Crystals

A new approach to optical monitors for gases is introduced using cholesteric liquid crystals doped with reactive chiral compounds. The approach is based on cholesteric pitch length changes caused by a change in helical twisting power (HTP) of the chiral dopants upon reaction with the analyte. The concept is demonstrated for monitoring carbon dioxide via reversible carbamate formation and for oxygen using the irreversible oxidation of a chiral dithiol to a disulfide. Monitoring of CO(2) was achieved by doping a commercial cholesteric liquid crystalline mixture (E7) with 1.6% mol of the 1:1 complex of an optically pure diamine with a TADDOL derivative. Upon exposure to carbon dioxide, the reflection band of a thin film of the mixture shifted from 637 to 495 nm as a consequence of dissociation of the complex after carbamate formation of the diamine. An O(2) monitor was obtained by doping E7 with a chiral binaphthyl dithiol derivative and a nonresponsive codopant. The reflection band of the oxygen monitor film changed from 542 to 600 nm, due to the conformational change accompanying oxidation of the dithiol to disulfide. These monitoring mechanisms hold promise for application in smart packaging, where carbon dioxide and oxygen are of special interest because of their roles in food preservation.

Ternary Protein Complex of Ferredoxin, Ferredoxin:Thioredoxin Reductase, and Thioredoxin Studied by Paramagnetic NMR Spectroscopy

In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.

The formation of disulfides by the [Fe(nta)Cl 2] 2− catalyzed air oxidation of thiols and dithiols

The complexes (cnt) 2[Fe(nta)Cl 2], where nta = nitrilotriacetate and cnt = Et 4N + or PyH +, catalyze the air oxidation of thiols to disulfides under ambient conditions. Dithiols are converted to linear and cyclic oligomers that differ in their terminal groups as a function of the counterion, cnt. Cysteine ethyl ester was converted to the corresponding cystine diethylester in high yield.

Generating disulfides enzymatically: Reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.

A Chemical Ground of de novo Protein Disulfide Bond Generation in the Cell

Many secretory proteins undergo oxidative folding, during which they acquire intra- or intermolecular disulfide bonds. In the periplasm of Escherichia coli, DsbA functions as a primary disulfide bond donor. DsbB, which is responsible for reoxidation of DsbA, acts as a molecular machine that transforms an oxidizing equivalent of ubiquinone into a protein disulfide. A similar oxidative system exists in the ER of eukaryotic cells, where Ero1p and FAD play a pivotal role in the disulfide bond creation. This review describes the reaction mechanism of ubiquinone-dependent dithiol oxidation in Escherichia coli and proposes its similarities to the eukaryotic FAD-dependent one.

Aromatic disulfide polymers back to macrocyclic disulfide oligomers via cyclo-depolymerization reaction

A brand new and highly efficient method has been developed for the synthesis of aromatic macrocyclic disulfide oligomers. Cyclization was achieved by cyclo-depolymerization (CDP) of aromatic disulfide polymer in the presence of catalytic amount of thiols and weak base. The cyclic oligomers undergo a facile free-radical ring-opening polymerization to form high molecular weight polymer. Comparing with the conventional method for the preparation of macrocyclic aromatic oligomers by catalytic oxidation of dithiols, highly dilute condition was not necessary using the new method. The macrocyclics in high yield and with narrow molecular weight distribution were obtained. This method also provides a convenient technology for the recycle of aromatic disulfide polymers. ▪

Catalysis of Protein Disulfide Bond Isomerization in a Homogeneous Substrate

Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a beta hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N and C terminus contain a fluorescence donor (tryptophan) and acceptor (N(epsilon)-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E(o') = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys-Gly-His-Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/K(M) = 1.7 x 10(5) M(-1) s(-1), which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.

Identification of an Entamoeba histolytica gene encoding a protein disulfide isomerase that functionally complements the dsbA mutation in Escherichia coli

0 d In vivo, as in vitro, the tertiary structure of a protein is etermined by its amino acid sequence. Despite the ability f some isolated polypeptide to be denatured and refolded n vitro in the absence of other proteins, correct folding and ssembly of polypeptides in vivo involves the assistance of ther proteins, collectively named “foldases” [1]. Protein isulfide isomerase activity (PDI, EC 5.3.4.1), encoded by n eukaryotic family of foldase genes, is a protein-thiol xidoreductase that catalyzes the oxidation, reduction, and somerization of disulfide bonds in nascent polypeptides [2]. n the endoplasmic reticulum compartment, PDI exhibits xidative and reductive activities, while in reducing envionments (such as the cytosol, endosomes or cytoplasmic embrane) behaves just as a reductase [3]. In yeast and ammalian cells, the PDI subcellular localization and funcion suggest that it plays a key roll in the secretory pathway 4]. Structurally, PDI proteins share a region, of about 00 amino acid residues, that is highly homologous to the rokaryotic protein thioredoxin, thus known as “thioredoxinr Tx-domain”. The active site of Tx-domains contains the otif WCGHCK, in which the two cysteine residues cycle Amebiasis is a major cause of death in developing countries. Worldwide, it has been estimated that at least 40,000 people die every year by invasive infection of the etiological agent, the protozoan parasite Entamoeba histolytica [5,6]. In this pathogen the formation of accurate intraor inter-molecular disulfide bonds is an important biochemical step in favor of the correct folding status of proteins, such as the virulence factors amoebapores and Gal/GalNAc-binding lectin [7,8]. We have investigated whether E. histolytica encodes proteins with disulfide oxidoreductase activity, like PDI enzymes, particularly with an active in vivo oxidase activity. Our approach was based on the molecular isolation of actively expressed genes, followed by a functional complementation test. In an initial report, we described the isolation of a cDNA fragment coding for the C-terminal region of a putative EhPDI protein [9]. Here we extend that observation and report the complete characterization of an E. histolytica cDNA sequence encoding a polypeptide with high similarity to the PDI protein family, hence named EhPDI, as well as a phylogenetic inference analysis, and the functional evaluation of its oxidase activity (using an etween the dithiol and disulfide oxidation states [2,4].