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PubMed1996

Enzyme-monitored turnover of Escherichia coli thioredoxin reductase: insights for catalysis.

Thioredoxin reductase from Escherichia coli is a member of the pyridine nucleotide-disulfide oxidoreductase family, and contains one FAD and one redox-active disulfide per subunit. It is known that two other well-studied members of this family, lipoamide dehydrogenase and glutathione reductase, cycle between the two electron-reduced and fully oxidized forms in catalysis. Enzyme-monitored turnover shows that the spectrum of thioredoxin reductase during turnover represents fully reduced flavin with NADP(H) bound. Whether the pyridine nucleotide bound is NADPH or NADP+ is dependent on the concentration of each species, i.e., how far turnover has progressed. It is also shown that the midpoint potentials of this enzyme are increased through the differential binding of NADP+ to the oxidized and reduced form of the enzyme. When combined with other kinetic and oxidation/reduction studies of this enzyme, these results indicate that thioredoxin reductase cycles between the four-electron-reduced and two-electron-reduced forms in catalysis, and that it does so with pyridine nucleotide bound. These results clarify the mechanism of thioredoxin reductase in relation to the known structure the enzyme, and provide support for earlier work in which we proposed that this enzyme utilizes a ternary complex mechanism in catalysis.

Catalysis↗
PubMed1998

Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis.

The flavoenzyme glutathione reductase catalyzes the NADPH-dependent reduction of glutathione disulfide, yielding two molecules of glutathione. The oxidation-reduction potentials, Eox/EH2 (two-electron reduced enzyme), for yeast, Escherichia coli, and human glutathione reductase have been determined between pH 6.0 and 9.8 relative to the nonphysiological substrate couple NAD+/NADH and were found to be -237, -243, and -227 mV (+/-5 mV) at pH 7.0 and 20 degreesC, respectively. The potential as a function of pH demonstrated slopes of -51, -45, and -42 mV/pH unit, respectively, at low pH and -37, -31, and -34 mV/pH unit, respectively, at high pH. The change in slope indicated pKa values of 7.4, 8.5, and 7.6, respectively. The slopes indicate that two protons are associated with the two-electron reduction of Eox at low pH and that only one proton is involved with the two-electron reduction of Eox at high pH, provided that the effects of nearby titratable residues are considered in the data analysis. The influence of four such groups, Cys50, Cys45, His456', and either Tyr107 or the flavin-(N3), has been included (residue numbering refers to the yeast sequence). The enzyme loses activity upon deprotonation of the acid-base catalyst at high pH. Since the pKa ascribed to the EH2-to-EH- ionization is lower than the pKa of the acid-base catalyst, both the EH2 and EH- forms of glutathione reductase must be catalytically active, in contrast to the closely related enzyme lipoamide dehydrogenase, for which only EH2 is active.

Catalysis↗
PubMed2005

RAR-related orphan receptor A isoform 1 (RORa1) is disrupted by a balanced translocation t(4;15)(q22.3;q21.3) associated with severe obesity.

We have identified a family comprising a mother and two children with idiopathic and profound obesity body mass index (BMI) 41-49 kg/m(2). The three family members carry a balanced reciprocal chromosome translocation t(4;15). We present here the clinical features of the affected individuals as well as the physical mapping and cloning of the chromosomal breakpoints. A detailed characterisation of the chromosomal breakpoints at chromosomes 4 and 15 revealed that the translocation is almost perfectly balanced with a very short insertion/deletion. The chromosome 15 breakpoint is positioned in intron 1 of the RAR-related orphan receptor A isoform 1 (RORa1) and the chromosome 4 breakpoint is positioned 133 kb telomeric to the transcriptional start of the unc-5 homolog B (UNC5C) and 154 kb centromeric of the transcriptional start of the pyruvate dehydrogenase (lipoamide) alpha 2 (PDHA2). The rearrangement creates a fusion gene, which includes the RORa1 exon 1 and UNC5C that is expressed in frame in adipocytes from the affected patients. We also show that this transcript is translated into a protein. From previous reports, it is shown that RORa1 is implicated in the regulation of adipogenesis and lipoprotein metabolism. We hypothesise that the obesity in this family is caused by (i) haploinsufficiency for RORa1 or, (ii) a gain of function mechanism mediated by the RORa1-UNC5C fusion gene.

Adipocytes↗
PubMed2004

Photosensitized NADH formation system with multilayer TiO2 film.

A TiO(2)/polymer film on a quartz plate fabricated by a layer-by-layer method was employed for NADH production from NAD(+) with lipoamide dehydrogenase and methyl viologen in Tris-HCl buffer on irradiation with UV light, and the ultra thin film prevented from enzyme deactivating effectively.

Buffers↗
PubMed2003

NarE: a novel ADP-ribosyltransferase from Neisseria meningitidis.

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.

ADP Ribose Transferases↗
PubMed2000

Thioredoxin reductase two modes of catalysis have evolved.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.

Animals↗
PubMed2004

S-nitroso proteome of Mycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense.

The immune response to Mycobacterium tuberculosis (Mtb) includes expression of nitric oxide (NO) synthase (NOS)2, whose products can kill Mtb in vitro with a molar potency greater than that of many conventional antitubercular agents. However, the targets of reactive nitrogen intermediates (RNIs) in Mtb are unknown. One major action of RNIs is protein S-nitrosylation. Here, we describe, to our knowledge, the first proteomic analysis of S-nitrosylation in a whole organism after treating Mtb with bactericidal concentrations of RNIs. The 29 S-nitroso proteins identified are all enzymes, mostly serving intermediary metabolism, lipid metabolism, and/or antioxidant defense. Many are essential or implicated in virulence, including defense against RNIs. For each of two target enzymes tested, lipoamide dehydrogenase and mycobacterial proteasome ATPase, S-nitrosylation caused enzyme inhibition. Moreover, endogenously biotinylated proteins were driven into mixed disulfide complexes. Targeting of metabolic enzymes and antioxidant defenses by means of protein S-nitrosylation and mixed disulfide bonding may contribute to the antimycobacterial actions of RNIs.

Animals↗
PubMed2000

Synthesis and characterization of selenotrisulfide-derivatives of lipoic acid and lipoamide.

Thiol-containing compounds, such as glutathione and cysteine, react with selenite under specific conditions to form selenotrisulfides. Previous studies have focused on isolation and characterization of intermolecular selenotrisulfides. This study describes the preparation and characterization of intramolecular selenotrisulfide derivatives of lipoic acid and lipoamide. These derivatives, after separation from other reaction products by reverse-phase HPLC, exhibit an absorbance maximum at 288 nm with an extinction coefficient of 1,500 M(-1) small middle dotcm(-1). The selenotrisulfide derivative of lipoic acid was significantly stable at or below pH 8.0 in contrast to several other previously studied selenotrisulfides. Mass spectral analysis of the lipoic acid and lipoamide derivatives confirmed both the expected molecular weights and also the presence of a single atom of selenium as revealed by its isotopic distribution. The selenotrisulfide derivative of lipoic acid was found to serve as an effective substrate for recombinant human thioredoxin reductase as well as native rat thioredoxin reductase in the presence of NADPH. Likewise, the lipoamide derivative was efficiently reduced by NADH-dependent bovine lipoamide dehydrogenase. The significant in vitro stability of these intramolecular selenotrisulfide derivatives of lipoic acid can serve as an important asset in the study of such selenium adducts as model selenium donor compounds for selenophosphate biosynthesis and as rate enhancement effectors in various redox reactions.

Animals↗
PubMed1997

Sulfide-quinone reductase from Rhodobacter capsulatus. Purification, cloning, and expression.

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.'.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa, exhibiting absorption and fluorescence spectra typical for a flavoprotein and similar to the SQR from the cyanobacterium Oscillatoria limnetica. From N-terminal and tryptic peptide sequences of the pure protein a genomic DNA clone was obtained by polymerase chain reaction amplification. Its sequence contains an open reading frame of 1275 base pairs (EMBL nucleotide sequence data base, accession no. X97478X97478) encoding the SQR of R. capsulatus. The deduced polypeptide consists of 425 amino acid residues with a molecular mass of 47 kDa and a net charge of +9. The high similarity (72%)/identity (48%) between the N termini of the cyanobacterial and the bacterial enzyme was confirmed and extended. Both enzymes exhibit the FAD/NAD(P) binding betaalphabeta-fold (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). The complete sequence of the SQR from R. capsulatus shows further similarity to flavoproteins, in particular glutathione reductase and lipoamide dehydrogenase. The cloned sqr was expressed in Escherichia coli in a functional form.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell. Recent studies revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases. The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. The putative essential amino acids at the catalytic center of large thioredoxin reductase from P. falciparum were determined by using site-directed mutagenesis techniques. To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues. Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine. All mutants were expressed in the E. coli system and characterized. Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity. These results clearly identify Cys88 and Cys93 as the active site thiols of large thioredoxin reductase. The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity. These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase. Spectral analysis of wild-type P. falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase. This spectral feature, recently also reported for the human placenta protein (Arscott, L. D., Gromer, S., Schirmer, R. H., Becker K., and Williams, C. H., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3621-3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to small E. coli thioredoxin reductase.

Animals↗
PubMed1998

Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2-.) production by biological systems. However, its validity as a O2-.-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2-.. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2-. has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2-. production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2-. in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 microM in all of the O2-.-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/ NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2-. production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/ xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2-.-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2-.. Thus, LDCL still appears to be a valid probe for detecting O2-. production by enzymatic and cellular sources.

Acridines↗
PubMed2000

Intersubunit interactions in Plasmodium falciparum thioredoxin reductase.

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M(r) and the low M(r) proteins. The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.

Animals↗
PubMed2003

Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export.

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed. Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.

Base Sequence↗
PubMed2004

Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response.

Glucose-stimulated insulin secretion is a multistep process dependent on beta-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters beta-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenase-dependent metabolism or a buffering of the NADH response by other metabolic mechanisms.

Animals↗
PubMed2004

Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis.

The lpdA (Rv3303c) gene from Mycobacterium tuberculosis encoding a new member of the flavoprotein disulfide reductases was expressed in Escherichia coli, and the recombinant LpdA protein was purified to homogeneity. LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP+ per monomer. Although annotated as a probable lipoamide dehydrogenase in M. tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis. The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site. Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone. Significant primary deuterium kinetic isotope effects were observed with [4S-2H]NADH establishing that the enzyme promotes transfer of the C4-proS hydride of NADH. The absence of an isotope effect with [4S-2H]NADPH, the low Km value of 0.5 microm for NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP+ (Ki approximately 6 nm) and 2'-phospho-ADP-ribose (Ki approximately 800 nm), demonstrate the high affinity of LpdA for 2'-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP+ rather than NADH/NAD+. Modeling of NADP+ in the active site revealed that LpdA achieves the high specificity for NADP+ through interactions involving the 2'-phosphate of NADP+ and amino acid residues that are different from those in glutathione reductase.

Amino Acid Sequence↗
PubMed1976

Formation of ethylenethiourea from 5,6-dihydro-3H-imidazo[2,1-c]-1,2,4-dithiazole-3-thione by microorganisms and reducing agents.

Microorganisms formed readily ethylenethiourea (ETU) from 5,6-dihydro-3H-imidazo[2,1-c]-1,2,4-dithiazole-3-thione (DIDT), a spontaneous decomposition product of ethylenebisdithiocarbamates. This conversion also takes place after addition of reducing compounds like cysteine, glutathione or ascorbic acid. It consists of two steps: reduction of the S-S bond of DIDT with subsequent release of CS2 to form ETU. DIDT was reduced by NADH in the presence of enzyme extracts from Pseudomonas fluorescens or Asperigillus niger, or by commercial glutathione reductase or lipoamide dehydrogenase. ETU was formed as a result of this enzymatic reduction. The flavin compounds FMN and FAD were also able to promote the reduction of DIDT by NADH.

Aspergillus niger↗