Solution structure and dynamics of the reduced and oxidized forms of the N-terminal domain of PilB from Neisseria meningitidis

The secreted form of the PilB protein was proposed to be involved in pathogen survival fighting against the defensive host's oxidative burst. PilB protein is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively. The N-terminal domain, which possesses a CXXC motif, was recently shown to regenerate in vitro the reduced forms of the methionine sulfoxide reductase domains of PilB from their oxidized forms, as does the thioredoxin 1 from E. coli, via a disulfide bond exchange. The thioredoxin-like N-terminal domain belongs to the cytochrome maturation protein structural family, but it possesses a unique additional segment (99)FLHE (102) localized in a loop. This segment covers one edge of the active site in the crystal structure of the reduced form of the N-terminal domain of PilB. We have determined the solution structure and the dynamics of the N-terminal domain from Neisseria meningitidis, in its reduced and oxidized forms. The FLHE loop adopts, in both redox states, a well-defined conformation. Subtle conformational and dynamic changes upon oxidation are highlighted around the active site, as well as in the FLHE loop. The functional consequences of the cytochrome maturation protein topology and those of the presence of FLHE loop are discussed in relation to the enzymatic properties of the N-terminal domain.     

The X-ray structure of the N-terminal domain of PILB from Neisseria meningitidis reveals a thioredoxin-fold

The secreted form of the PilB protein was recently shown to be bound to the outer membrane of Neisseria gonorrhoeae and proposed to be involved in survival of the pathogen to the host's oxidative burst. PilB is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase (Msr) A and B activities respectively, i.e. the ability to reduce specifically the S and the R enantiomers of the sulfoxide function of the methionine sulfoxides, which are easily formed upon oxidation of methionine residues. The N-terminal domain of PilB (Dom1(PILB)) of N.meningitidis, which possesses a CXXC motif, was recently shown to recycle the oxidized forms of the PilB Msr domains in vitro, as the Escherichia coli thioredoxin (Trx) 1 does. The X-ray structure of Dom1(PILB) of N.meningitidis determined here shows a Trx-fold, in agreement with the biochemical properties of Dom1(PILB). However, substantial structural differences with E.coli Trx1 exist. Dom1(PILB) displays more structural homologies with the periplasmic disulfide oxidoreductases involved in cytochrome maturation pathways in bacteria. The active site of the reduced form of Dom1(PILB) reveals a high level of stabilization of the N-terminal catalytic cysteine residue and a hydrophobic environment of the C-terminal recycling cysteine in the CXXC motif, consistent with the pK(app) values measured for Cys67 (<6) and Cys70 (9.3), respectively. Compared to cytochrome maturation disulfide oxidoreductases and to Trx1, one edge of the active site is covered by four additional residues (99)FLHE(102). The putative role of the resulting protuberance is discussed in relation to the disulfide reductase properties of Dom1(PILB).     

A structural analysis of the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product—methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.     

Thermodynamic aspects of DsbD-mediated electron transport

DsbD from Escherichia coli transports electrons from cytoplasmic thioredoxin across the inner membrane to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of three domains: a periplasmic N-terminal domain, a central transmembrane domain (tmDsbD) and a periplasmic C-terminal domain. Each domain contains two essential cysteine residues that are required for electron transport. In contrast to the quinone reductase DsbB, HPLC analysis of the methanol/hexane extracts of purified DsbD revealed no presence of quinones, suggesting that the tmDsbD interacts with thioredoxin and the periplasmic C-terminal domain exclusively via disulfide exchange. We also demonstrate that a DsbD variant containing only the redox-active cysteine pair C163 and C285 in tmDsbD, reconstituted into liposomes, has a redox potential of − 0.246 V. The results show that all steps in the DsbD-mediated electron flow are thermodynamically favorable.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.     

E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide

The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low K(M) value of 10 microM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in k(cat)/K(MetSO). Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.     

Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis

AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.     

Activity of one of two engineered heterodimers of AhpF, the NADH:peroxiredoxin oxidoreductase from Salmonella typhimurium, reveals intrasubunit electron transfer between domains

AhpF, the flavoprotein reductase component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the reduction of an intersubunit disulfide bond in the peroxidatic active site of the system's other component, AhpC, a member of the peroxiredoxin family. Previous studies have shown that AhpF can be dissected into two functional units, a thioredoxin reductase-like C-terminus (containing FAD and a redox-active disulfide, Cys345-Cys348) and an N-terminal domain containing a second redox-active disulfide center (Cys129-Cys132). The role of the N-terminal domain as the direct reductant of AhpC, mediating electron transfer from the C-terminal redox centers of AhpF, has been firmly established by several approaches. Not known, however, was whether the transfer of electrons between the C-terminal and N-terminal disulfide centers occurred as an inter- or intrasubunit process in dimeric AhpF. Two heterodimeric AhpF species were therefore created in which one of the two pathways was completely disrupted while the other was left partially intact in each construct. Only the heterodimer containing one monomer of wild type AhpF and a monomer of mutated (and truncated) AhpF exhibited peroxidase activity with AhpC indicating that electron transfer between domains of AhpF is an intrasubunit process.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Kinetic characterization of the catalytic mechanism of methionine sulfoxide reductase B from Neisseria meningitidis

Methionine sulfoxide reductases catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The methionine sulfoxide reductases family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the sulfoxide function. Both enzymes are monomeric and share a similar three-step chemical mechanism. First, in the reductase step, a sulfenic acid intermediate is formed with a concomitant release of 1 mol of methionine per mol of enzyme. Then, an intradisulfide bond is formed. Finally, Msrs return back to reduced forms via reduction by thioredoxin. In the present study, it is shown for the Neisseria meningitidis MsrB that (1) the reductase step is rate-determining in the process leading to formation of the disulfide bond and (2) the thioredoxin-recycling process is rate-limiting. Moreover, the data suggest that within the thioredoxin-recycling process, the rate-limiting step takes place after the two-electron chemical exchange and thus is associated with the release of oxidized thioredoxin.     

Solution structure and backbone dynamics of the cysteine 103 to serine mutant of the N-terminal domain of DsbD from Neisseria meningitidis

The DsbD protein is essential for electron transfer from the cytoplasm to the periplasm of Gram-negative bacteria. Its N-terminal domain dispatches electrons coming from cytoplasmic thioredoxin (Trx), via its central transmembrane and C-terminal domains, to its periplasmic partners: DsbC, DsbE/CcmG, and DsbG. Previous structural studies described the latter proteins as Trx-like folds possessing a characteristic C-X-X-C motif able to generate a disulfide bond upon oxidation. The Escherichia coli nDsbD displays an immunoglobulin-like fold in which two cysteine residues (Cys103 and Cys109) allow a disulfide bond exchange with its biological partners.We have determined the structure in solution and the backbone dynamics of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis. Our results highlight significant structural changes concerning the beta-sheets and the local topology of the active site compared with the oxidized form of the E. coli nDsbD. The structure reveals a "cap loop" covering the active site, similar to the oxidized E. coli nDsbD X-ray structure. However, regions featuring enhanced mobility were observed both near to and distant from the active site, revealing a capacity of structural adjustments in the active site and in putative interaction areas with nDsbD biological partners. Results are discussed in terms of functional consequences.     

The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat

The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.     

Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.     

Structure of the translocator domain of a bacterial autotransporter

Autotransporters are virulence-related proteins of Gram-negative bacteria that are secreted via an outer-membrane-based C-terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N-terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro-folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12-stranded beta-barrel with a hydrophilic pore of 10 x 12.5 A that is filled by an N-terminal alpha-helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger-domain transport through the hydrophilic channel within the beta-barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger-domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer-membrane proteins, on which autotransporters are dependent.     

Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures

A cDNA encoding a plant-type APS reductase was isolated from an axenic cell suspension culture of Catharanthus roseus (Genbank/EMBL-databank accession number U63784). The open reading frame of 1392 bp (termed par) encoded for a protein (Mr=51394) consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase. The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa. The homologous protein from pea chloroplast stroma was detected using anti:par polyclonal antibodies. To investigate the catalytical function of the different domains deleted par proteins were purified. ParDelta1 lacking the transit sequence liberated sulfite from APS (Km 2.5+/-0.23 microM) in vitro with glutathione (Km 3+/-0.64 mM) as reductant (Vmax 2.6+/-0.14 U mg-1, molecular activity 126 min-1). ParDelta2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (Km 15. 3+/-1.27 microM, Vmax 0.6+/-0.014 U mg-1). Glutaredoxin, GSH or DTT were ineffective substitutes. ParDelta1 (35.4%) and parDelta2 (21. 8%) both exhibited insulin reductase activity comparable to thioredoxin (100%). Protein disulfide isomerase activity was observed for parDelta1.     

Structural basis and kinetics of DsbD-dependent cytochrome c maturation

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.     

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.     

The activity of barley NADPH-dependent thioredoxin reductase C is independent of the oligomeric state of the protein: tetrameric structure determined by cryo-electron microscopy

Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H(2)O(2). These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.