Pyridine Nucleotide-disulfide Oxidoreductase Research Articles

The "Doorstop Pocket" In Thioredoxin Reductases─An Unexpected Druggable Regulator of the Catalytic Machinery.

Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP+ during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.

Modulation in gene expression and enzyme activity suggested the roles of monodehydroascorbate reductase in development and stress response in bread wheat

Monodehydroascorbate reductase (MDHAR) is a crucial enzymatic antioxidant of the ascorbate-glutathione pathway involved in reactive oxygen species scavenging. Herein, we identified 15 TaMDHAR genes in bread wheat. Phylogenetic analysis revealed their clustering into three groups, which are also related to the subcellular localization in the peroxisome matrix, peroxisome membrane, and chloroplast. Each TaMDHAR protein consisted of two conserved domains; Pyr_redox and Pyr_redox_2 of the pyridine nucleotide disulfide oxidoreductase family. The occurrence of diverse groups of cis-regulatory elements in the promoter region and their interaction with numerous transcription factors suggest assorted functions of TaMDHARs in growth and development and in light, phytohormones, and stress responses. Expression analysis in various tissues further revealed their importance in vegetative and reproductive development. In addition, the differential gene expression and enhanced enzyme activity during drought, heat, and salt treatments exposed their role in abiotic stress response. Interaction of MDHARs with various antioxidant enzymes and biochemicals related to the ascorbate-glutathione cycle exposed their synchronized functioning. Interaction with auxin indicated the probability of cross-talk between antioxidants and auxin signaling. The miR168a, miR169, miR172 and others interaction with various TaMDHARs further directed their association with developmental processes and stress responses. The current study provides extensive information about the importance of TaMDHARs, moreover, the precise role of each gene needs to be established in future studies.

Purification and Characterization of Glutathione Reductase Enzyme from Arum Maculatum Leaf

Arum species grow in temperate and Mediterranean climates and have been used for hundreds of years for food and medicinal purposes, although they are highly toxic if not cooked using proper techniques. Glutathione reductase (GR) is a member of the pyridine nucleotide disulfide oxidoreductase family of flavoenzymes that catalyzes the reduction of glutathione disulfide (GSSG) to reduced GSH using NADPH or NADH. In this study, GR enzyme was characterized by partial purification processes including homogenate preparation, ammonium sulfate precipitation and dialysis from the leaf of Arum maculatum plant. The highest enzyme activity was found at 40-60% saturation range. Optimum ionic strength, pH and substrate concentration were investigated for GR enzyme from A. maculatum leaf. As a result of the study, these values were found to be 150 mM potassium phosphate buffer, pH 7.00, and 0.18 mM, respectively. The GR enzyme was partially purified from the leaf of the A. maculatum with a specific activity of 1.640 EU mg-1 in 34.9% yield, 1.108-fold. This study is the first study in terms of purification and characterization of GR enzyme from A. maculatum leaf.

Crosstalk between ROCK1 and PYROXD1 regulates CAFs activation and promotes laryngeal squamous cell carcinoma metastasis

We previously found that the Rho-associated kinase 1 (ROCK1) activated Cancer-associated fibroblasts (CAFs) to promote LSCC metastasis. Accumulating evidence indicates that pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1) is an oncogene; however, the crosstalk between ROCK1 and PYROXD1 in LSCC metastasis remains largely unknown. Here, we found that ROCK1 could target PYROXD1. The knockdown of ROCK1 expression reduces the expression of PYROXD1, while the knockdown of PYROXD1 expression did not alter the expression of ROCK1 indicating that ROCK1 is upstream of PYROXD1. Further, LSCC cells cocultured with PYROXD1 knocked-down CAFs exhibited lower proliferation, migration, invasion and metastasis abilities. Conversely, LSCC cells cocultured with PYROXD1-overexpressing CAFs showed opposite results. In conclusion, the crosstalk between ROCK1 and PYROXD1 regulated CAFs activation and promoted LSCC metastasis.

Exploration of glutathione reductase for abiotic stress response in bread wheat (Triticum aestivum L.)

A total of seven glutathione reductase (GR) genes were identified in Triticum aestivum, which were used for comparative structural characterization, phylogenetic analysis and expression profiling with the GR genes of other cereal plants. The modulated gene expression and enzyme activity revealed the role of GRs in abiotic stress response in T. aestivum. Glutathione reductase (GR) is an enzymatic antioxidant that converts oxidized glutathione (GSSG) into reduced glutathione (GSH) through the ascorbate-glutathione cycle. In this study, a total of seven GR genes forming two homeologous groups were identified in the allohexaploid genome of bread wheat (Triticum aestivum). Besides, we identified three GR genes in each Aegilops tauschii, Brachypodium distachyon, Triticum urartu and Sorghum bicolor, which were used for comparative characterization. Phylogenetic analysis revealed the clustering of GR proteins into two groups; class I and class II, which were predicted to be localized in cytoplasm and chloroplast, respectively. The exon-intron and conserved motif patterns were almost conserved in each group, in which a maximum of 10 and 17 exons were present in chloroplastic and cytoplasmic GRs, respectively. The protein structure analysis confirmed the occurrence of conserved pyridine nucleotide disulfide oxidoreductase (Pyr_redox) and pyridine nucleotide disulfide oxidoreductase dimerization (Pyr_redox_dim) domains in each GR. The active site of GR proteins consisted of two conserved cysteine residues separated by four amino acid residues. Promoter analysis revealed the occurrence of growth and stress-related cis-active elements. Tissue-specific expression profiling suggested the involvement of GRs in both vegetative and reproductive tissue development in various plants. The differential expression of TaGR genes and enhanced GR enzyme activity suggested their roles under drought, heat, salt and arsenic stress. Interaction of GRs with other proteins and chemical compounds of the ascorbate-glutathione cycle revealed their coordinated functioning. The current study will provide a foundation for the validation of the precise role of each GR gene in future studies.

Clinical and genetic characterization of PYROXD1-related myopathy patients from Turkey.

Congenital myopathies (CMs) are a heterogeneous group of inherited muscle disorders characterized by muscle weakness at birth, while limb-girdle muscular dystrophies (LGMD) have a later onset and slower disease progression. Thus, detailed clinical phenotyping of genetically defined disease entities are required for the full understanding of genotype-phenotype correlations. A recently defined myopathic genetic disease entity is caused by bi-allelic variants in a gene coding for pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1) with unknown substrates. Here, we present three patients from two consanguineous Turkish families with mild LGMD, facial weakness, normal CK levels, and slow progress. Genomic analyses revealed a homozygous known pathogenic missense variant (c.464A>G, p.Asn155Ser) in family 1 with two affected females. In the affected male of family 2, we found this variant in a compound heterozygous state together with a novel frameshift variant (c.329_332delTCTG, p.Leu112Valfs*8), which is the second frameshift variant known so far in PYROXD1. We have been able to define a large homozygous region in family 1 sharing a common haplotype with family 2 in the critical region. Our data suggest that c.464A>G is a Turkish founder mutation. To gain deeper insights, we performed a systematic review of all published PYROXD1-related myopathy cases. Our analysis showed that the c.464A > G variant was found in 87% (20/23) of the patients and that it may cause either a childhood- or adult-onset phenotype, irrespective of its presence in a homozygous or compound heterozygous state. Interestingly, only four patients had elevated CK levels (up to 1000 U/L), and cardiac involvement was found in few compound heterozygous cases.

Mutational studies on Leishmania donovani dihydrolipoamide dehydrogenase (LdBPK291950.1) indicates that the enzyme may not be classical class-I pyridine nucleotide-disulfide oxidoreductase

We report biochemical studies on two Cys residues mutation (Cys15Thr, Cys38Gly) nearest to the active site and three other amino acid substitution mutations expected to be the part of active site of LdDLDH_Variant1. Our biochemical studies show that the replacement of Cys15 increases the Km for dihydrolipoamide (DLD) substrate by five folds and NAD+ by three fold indicating that this mutation affects the binding of DLD and NAD+ significantly. Cys38 was also mutated to ‘Gly’ which resulted in nine fold greater Km for NAD+ without affecting Km for DLD. However, even after these mutations (Cys15Thr and Cys38Gly), reduced enzyme activity suggests that both the ‘Cys’ residues are not involved in disulfide bond formation but affect the binding of substrates. The data hints towards the possibility of a different catalytic mechanism from the classical class I - pyridine nucleotide-disulfide oxidoreductase. Remaining other mutated residues Ala48Ile, Asp49Gly, and Ala54Ile showed an increase in two to three-folds Km value for NAD+, which means these residues are important for the binding of NAD+ to the enzyme. However, Ala48Ile and Asp49Gly mutations showed a decrease of Km for DLD. Apart from the mutational studies, localization of LdDLDH_Variant2 of LdDLDH was also analyzed.

Protection of the transplant kidney during cold perfusion with doxycycline: proteomic analysis in a rat model

BackgroundIt has been previously shown that doxycycline (Doxy) protects the kidney from preservation injury by inhibition of matrix metalloproteinase. However, the precise molecular mechanism involved in this protection from injury is not known. We used a pharmaco-proteomics approach to identify potential molecular targets associated with kidney preservation injury.MethodsRat kidneys were cold perfused with or without doxycycline (Doxy) for 22 h. Kidneys perfusates were analyzed for the presence of injury markers such as lactate dehydrogenase (LDH), and neutrophil-gelatinase associated lipocalin (NGAL). Proteins extracted from kidney tissue were analyzed by 2-dimensional gel electrophoresis. Proteins of interest were identified by mass spectrometry.ResultsTriosephosphate isomerase, PGM, dihydropteridine reductase-2, pyridine nucleotide-disulfide oxidoreductase, phosphotriesterase-related protein, and aminoacylase-1A were not affected by cold perfusion. Perfusion with Doxy increased their levels. N(G),N(G)-dimethylarginine dimethylaminohydrolase and phosphoglycerate kinase 1 were decreased after cold perfusion. Perfusion with Doxy led to an increase in their levels.ConclusionsThis study revealed specific metabolic enzymes involved in preservation injury and in the mechanism whereby Doxy protects the kidney against injury during cold perfusion.

Characterization of recombinant glutathione reductase from Antarctic yeast Rhodotorula mucilaginosa

Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide, which helps to maintain a cellular reducing environment during stress in organisms. However, GR of polar yeast has not been well-characterized so far. To fully understand the molecular and enzymatic properties of GR from extreme area and broaden its knowledge, a cDNA-encoding GR from Antarctic sea-ice yeast Rhodotorula mucilaginosa (designated as RmGR) was cloned and expressed in Escherichia coli. The open reading frame of RmGR was 1500 bp, encoding an enzyme of 499 amino acids with a predicted pI of 6.07 and molecular weight of 54.8 kDa. SDS-PAGE and gel filtration analysis results showed that the RmGR was a homodimer. Conserved sequence analysis revealed that RmGR behaved typical characteristics of GR, containing a pyridine nucleotide-disulfide oxidoreductase active site, a flavin adenine dinucleotide (FAD), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs and two glutathione oxidized binding motifs. The recombinant enzyme displayed relatively high stability at pH 3.0–6.0 and 20–40 °C with optimal enzymatic activity at 30 °C and pH 7.5. Real-time quantitative PCR data showed that the expression of RmGR gene was upregulated after copper treatment of yeast cells. Moreover, RmGR-transformed E. coli exhibited stronger growth profiles than cells with an empty vector after copper and cadmium treatment. Our results demonstrated the possible function of RmGR in adaptation to heavy metals and its potential application in heterologous expression systems.

A siRNA-based method for efficient silencing of PYROXD1 gene expression in the colon cancer cell line HCT116.

The aim of this study was to determine the biological function of pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1), a recently discovered protein, in colon cancer cell line HCT116. The small interfering RNA (siRNA) was designed rationally on the basis of the target sequence against PYROXD1. Relative PYROXD1 mRNA levels were measured by aquantitative real-time polymerase chain reaction. Flow cytometry was performed to monitor tumor cells proliferation and apoptosis after siRNA transfection. Knockdown of PYROXD1arrested the cell cycle, and induced late apoptosis in colon cancer cell line HCT116 DISCUSSION: Taken together, these results revealed the critical roles of PYROXD1 in regulating cell cycle and apoptosis and possibly will signify its therapeutic potential for targeting colorectal cancer models.

Potential for Heightened Sulfur-Metabolic Capacity in Coastal Subtropical Microalgae

SummaryThe activities of microalgae support nutrient cycling that helps to sustain aquatic and terrestrial ecosystems. Most microalgal species, especially those from the subtropics, are genomically uncharacterized. Here we report the isolation and genomic characterization of 22 microalgal species from subtropical coastal regions belonging to multiple clades and three from temperate areas. Halotolerant strains including Halamphora, Dunaliella, Nannochloris, and Chloroidium comprised the majority of these isolates. The subtropical-based microalgae contained arrays of methyltransferase, pyridine nucleotide-disulfide oxidoreductase, abhydrolase, cystathionine synthase, and small-molecule transporter domains present at high relative abundance. We found that genes for sulfate transport, sulfotransferase, and glutathione S-transferase activities were especially abundant in subtropical, coastal microalgal species and halophytic species in general. Our metabolomics analyses indicate lineage- and habitat-specific sets of biomolecules implicated in niche-specific biological processes. This work effectively expands the collection of available microalgal genomes by ∼50%, and the generated resources provide perspectives for studying halophyte adaptive traits.

Deletion of BmoR affects the expression of genes related to thiol/disulfide balance in Bacteroides fragilis

Bacteroides fragilis, an opportunistic pathogen and commensal bacterium in the gut, is one the most aerotolerant species among strict anaerobes. However, the mechanisms that control gene regulation in response to oxidative stress are not completely understood. In this study, we show that the MarR type regulator, BmoR, regulates the expression of genes involved in the homeostasis of intracellular redox state. Transcriptome analysis showed that absence of BmoR leads to altered expression in total of 167 genes. Sixteen of these genes had a 2-fold or greater change in their expression. Most of these genes are related to LPS biosynthesis and carbohydrates metabolism, but there was a significant increase in the expression of genes related to the redox balance inside the cell. A pyridine nucleotide-disulfide oxidoreductase located directly upstream of bmoR was shown to be repressed by direct binding of BmoR to the promoter region. The expression of two other genes, coding for a thiosulphate:quinone-oxidoreductase and a thioredoxin, are indirectly affected by bmoR mutation during oxygen exposure. Phenotypic assays showed that BmoR is important to maintain the thiol/disulfide balance in the cell, confirming its relevance to B. fragilis response to oxidative stress.

A broader active site in Pyrococcus horikoshii CoA disulfide reductase accommodates larger substrates and reveals evidence of subunit asymmetry.

Within the family of pyridine nucleotide disulfide oxidoreductase (PNDOR), enzymes are a group of single‐cysteine containing FAD‐dependent reductases that utilize a tightly bound coenzyme A to assist in the NAD(P)H‐dependent reduction of di‐, per‐, and polysulfide substrates in bacteria and archaea. For many of these homodimeric enzymes, it has proved difficult to determine the substrate specificity and metabolic function based on sequence and genome analysis alone. Coenzyme A‐disulfide reductase (CoADR) isolated from Pyrococcus horikoshii (phCoADR) reduces Co‐A per‐ and polysulfides, but, unlike other highly homologous members of this group, is a poor CoA disulfide reductase. The phCoADR structure has a narrower access channel for CoA substrates, which suggested that this restriction might be responsible for the enzyme's poor activity toward the bulky CoA disulfide substrate. To test this hypothesis, the substrate channel was widened by making four mutations along the channel wall (Y65A, Y66A, P67G, and H367G). The structure of the quadruple mutant shows a widened substrate channel, which is supported by a fourfold increase in k cat for the NAD(P)H‐dependent reduction of CoA disulfide and enhanced activity toward the substrate at lower temperatures. Anaerobic titrations of the enzyme with NADH revealed a half‐site reactivity not observed with the wild‐type enzyme in which one subunit of the enzyme could be fully reduced to an EH4 state, while the other remained in an EH2 or EH2·NADH state. These results suggest that for these closely related enzymes, substrate channel morphology is an important determinant of substrate specificity, and homology modeling will be the preferred technique for predicting function among PNDORs.

Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F

The ability of the vancomycin-resistant Enterococcus faecalis (V583) to restore redox homeostasis via antioxidant defense mechanism is of importance, and knowledge into this defense is essential to understand its antibiotic-resistance and survival in hosts. The flavoprotein disulfide reductase AhpR, composed of the subunits AhpC and AhpF, represents one such vital part. Circular permutation was found to be a feature of the AhpF protein family. E. faecalis (V583) AhpF (EfAhpF) appears to be a representative of a minor subclass of this family, the typically N-terminal two-fold thioredoxin-like domain (NTD_N/C) is located at the C-terminus, whereas the pyridine nucleotide-disulfide oxidoreductase domain is encoded in the N-terminal part of its sequence. In EfAhpF, these two domains are connected via an unusually long linker region providing optimal communication between both domains. EfAhpF forms a dimer in solution similar to Escherichia coli AhpF. The crystallographic 2.3Å resolution structure of the NTD_N/C domain reveals a unique loop-helix stretch (409ILKDTEPAKELLYGIEKM426) not present in homologue domains of other prokaryotic AhpFs. Deletion of the unique 415PAKELLY421-helix or of 415PAKELL420 affects protein stability or attenuates peroxidase activity. Furthermore, mutation of Y421 is described to be essential for E. faecalis AhpF's optimal NADH-oxidative activity.

Modulation of the flavin-protein interactions in NADH peroxidase and mercuric ion reductase: a resonance Raman study.

NADH peroxidase (Npx) and mercuric ion reductase (MerA) are flavoproteins belonging to the pyridine nucleotide:disulfide oxidoreductases (PNDO) and catalyzing the reduction of toxic substrates, i.e., hydrogen peroxide and mercuric ion, respectively. To determine the role of the flavin adenine dinucleotide (FAD) in the detoxification mechanism, the resonance Raman (RR) spectra of these enzymes under various redox and ligation states have been investigated using blue and/or near-UV excitation(s). These data were compared to those previously obtained for glutathione reductase (GR), another enzyme of the PNDO family, but catalyzing the reduction of oxidized glutathione. Spectral differences have been detected for the marker bands of the isoalloxazine ring of Npx, MerA, and GR. They provide evidence for different catalytic mechanisms in these flavoproteins. The RR modes of the oxidized and two-electron reduced (EH2) forms of Npx are related to very tight flavin-protein interactions maintaining a nearly planar conformation of the isoalloxazine tricycle, a low level of H-bonding at the N1/N5 and O2/O4 sites, and a strong H-bond at N3H. They also indicate minimal changes in FAD structure and environment upon either NAD(H) binding or reduction of the sulfinic redox center. All these spectroscopic data support an enzyme functioning centered on the Cys-SO-/Cys-S- redox moiety and a neighbouring His residue. On the contrary, the RR data on various functional forms of MerA are indicative of a modulation of both ring II distortion and H-bonding states of the N5 site and ring III. The Cd(II) binding to the EH2-NADP(H) complexes, biomimetic intermediates in the reaction of Hg(II) reduction, provokes important spectral changes. They are interpreted in terms of flattening of the isoalloxazine ring and large decreases in H-bonding at the N5 site and ring III. The large flexibility of the FAD structure and environment in MerA is in agreement with proposed mechanisms involving C4a(flavin) adducts.

Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (Haliotis discus discus): Insights as a potent player in host antioxidant defense

Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H2O2, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H2O2. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171–246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu+2 and Cd+2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.

Non-innocent ligands in copper complexes catalyzed oxidation of thiols

ABSTRACT Copper(II) chloride complexes with nitrogen-containing ligands (amines, amides, heterocycles) were studied as catalysts of selective oxidation of thiols to disulfides. This process has an important biochemical analogue – the formation of disulfide bridge in proteins [Wang C, Wesener SR, Zhang H, Cheng Y-Q. An FAD-dependent pyridine nucleotide-disulfide oxidoreductase is involved in disulfide bond formation in FK228 anticancer depsipeptide. Chem and Biol. 2009;16:585–593]; which affects their activity [Zhang L, Chou CP, Moo-Young M. Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system. Biotech Adv. 2011;29:923–929]. Also, thiol oxidation is an important process of oil sweetening. The search for effective and stable catalysts for this reaction is still topical [Ganguly SK, Das G, Kumar S, Sain B, Garg MO. Mechanistic kinetics of catalytic oxidation of 1-butanethiol in light oil sweetening. Catal Tod. 2012;198:246–251]. This article describes a mechanistic study of thiol oxidation and non-innocent ligands transformations during this reaction. The studied ligands are capable of oxidizing thiols, the reaction being followed by reoxidation of the reduced forms of the ligands with air oxygen. The oxidative activity of the ligand correlates with catalyst activity for thiol oxidation.

Investigating the Half‐site Reactivity of a Member of the PNDOR Family of Proteins

Half‐site reactivity is an enzyme characteristic in which the active sites of a multimeric enzyme “fire” alternately, which means that one‐half of the identical subunits is active at any one time (1). Many metabolically important enzymes, such as alcohol dehydrogenase (ADH) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), are known to exhibit half‐site reactivity (2). Two models, induced‐fit (or alternating sites cooperativity model) and preexistent asymmetry, were proposed in early literature to explain this half‐site reactivity (3). The pyridine nucleotide disulfide oxidoreductases (PNDORs) are members of a flavoprotein family that use NADH/NADPH as their reductive substrate, with most members of the PNDOR family being found in the form of homodimers. It has previously been observed that members of the PNDOR family show half‐site reactivity (via an alternating sites cooperativity model), as evidenced by subunit asymmetry (4,5): when their bound flavin coenzyme is reduced by NAD(P)H, only one subunit reduces, with the other subunit remaining oxidized. Recent data from our laboratory suggests that the CoADR from Pyrococcus horikoshii, a member of the PNDOR family, also shows half‐site reactivity.Although many enzymes with half‐site reactivity have been characterized kinetically and structurally, the cause of half‐site reactivity and its effect on enzymatic catalytic efficiency and mechanism remains unclear. We have designed asymmetrical dimers of a member of the PNDOR family which disrupts the ability of the subunits to react in an alternating fashion. Preliminary kinetic data suggests that in an asymmetrical dimer, in which one subunit has been inactivated by mutation of its redox‐active cysteine (with the other subunit remaining active), the phCoADR actually exhibits a 3‐fold increase in catalytic efficiency when compared to the wild‐type enzyme for a range of different substrates. In addition, a higher background oxidase activity is observed in the asymmetrical dimer. Our results suggest that subunit communication, in the induced fit model of half‐site reactivity, appears to slow the overall rate of the enzyme, perhaps acting as a control on enzyme activity. The mechanisms of this control will be discussed.Support or Funding InformationThis project is supported by NSF award MCB 1518306.

Purification and characterization of mitochondrial thioredoxin reductase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the in vitro effects of some metal ions on the enzyme

Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases. In this study, mitochondrial TrxR enzyme was purified from rainbow trout mitochondria. Thanks to the 2 consecutive procedures (preparation of homogenate and 2',5'-ADP Sepharose 4B affinity chromatography), the enzyme, having the specific activity of 11.9 EU mg protein-1, was purified with a yield of 2.38{\%} and 672-fold. The purity of the enzyme was monitored and the molecular weight of its subunits was calculated as 70 kDa by SDS-PAGE. The native molecular mass of the enzyme was found to be approximately 151 kDa by gel filtration chromatography. Characteristic and kinetic properties of the enzyme were also determined. Furthermore, Se$^{4+}$, Cu$^{2+}$, Co$^{2+}$, Ni$^{2+}$, Fe$^{3+}$, and Al$^{3+}$ metal ions' in vitro effects on mitochondrial TrxR purified from rainbow trout was investigated. While Se$^{4+}$ ion increased the enzyme activity, all of the other metal ions used in this study showed an inhibitory effect.

Metal- and Semimetal-Containing Inhibitors of Thioredoxin Reductase as Anticancer Agents.

The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide disulfide oxidoreductases playing a central role in cellular redox homeostasis and signaling pathways. Recently, these selenoproteins have emerged as promising therapeutic targets for anticancer drug development, often being overexpressed in tumor cells and contributing to drug resistance. Herein, we summarize the current knowledge on metal- and semimetal-containing molecules capable of hampering mammalian TrxRs, with an emphasis on compounds reported in the last decade.