Photoactivation of NADP-Malate Dehydrogenase by a Heterogeneous Photochemical System in Zea mays

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction in order to activate the NADP-malate dehydrogenase in light. The photocatalyst acridine orange or proflavin sensitized EDTA-dependent reduction of ferredoxin. In a complete system, consisting of a dye donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified NADP-MDH was observed in vitro. The chloroplast mediated redox activation of enzyme essentially required ferredoxin, while heterogeneous photochemical mediated activation of enzyme need not require ferredoxin. The heterogeneus photochemical system activated NADP-MDH by eight fold similar to chloroplasts mediated ferredoxin dependent redox activation but was not affected by the presence of disalicylinden propanediamine-1, 2-disulphonic acid while there was complete inhibition of chloroplasts mediated activation of NADP-MDH in presence of this inhibitor. These observations suggest that a thiol mediator is essential for reductive activation of NADP-MDH and ferredoxin is not required for photochemical activation.     

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.     

Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase.

Selenium compounds like selenite (SeO3(2-) may form a covalent adduct with glutathione (GSH) in the form of selenodiglutathione (GS-Se-SG), which is assumed to be important in the metabolism of selenium. We have isolated GS-Se-SG and studied its reactions with NADPH and thioredoxin reductase from calf thymus or with thioredoxin reductase and thioredoxin from Escherichia coli. Incubation of 0.1 microM calf thymus thioredoxin reductase or 0.1 microM thioredoxin reductase and 1 microM thioredoxin from E. coli with 5, 10, or 20 microM GS-Se-SG resulted in a fast initial reaction, followed by a large and continued oxidation of NADPH. However, anaerobic incubation of 0.1 microM calf thymus thioredoxin reductase and 20 microM GS-Se-SG resulted only in oxidation of a stoichiometric amount of NADPH; admission of oxygen started continuous NADPH oxidation. Contrary to the mammalian enzyme, GS-Se-SG was not a substrate for thioredoxin reductase from E. coli. The rate of the oxygen-dependent reaction between calf thymus thioredoxin reductase and GS-Se-SG was increased 2-fold in the presence of 4 mM GSH, indicating that HSe- was the reactive intermediate. Glutathione reductase from rat liver reduced GS-Se-SG with a very slow continued oxidation of NADPH, and the presence of the enzyme did not affect the oxygen-dependent nonstoichiometric oxidation of NADPH by GS-Se-SG and thioredoxin reductase. Fluorescence spectroscopy showed GS-Se-SG to be a very efficient oxidant of reduced thioredoxin from E. coli and kinetically superior to insulin disulfides. Thioredoxin-dependent reduction of CDP to dCDP by ribonucleotide reductase was effectively inhibited by GS-Se-SG.     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes.

A new over-expression system has been set up for Escherichia coli thioredoxin, yielding 55 mg purified protein/10 g fresh cells. This system has been used to produce thioredoxin modified by site-directed mutagenesis. Taking advantage of the structural and enzymatic similarity between E. coli and spinach m-type thioredoxin, Asp61 of E. coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes. The modification did not significantly alter the structure of the protein. Neither the rate of reduction of insulin and 5,5'-dithio-bis(2-nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH-dependent thioredoxin reductase, have been modified. The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin-dependent thioredoxin reductase in a light-activation reconstituted chloroplast system. The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose-1,6-bisphosphatase activation by the dithiothreitol-reduced thioredoxin, and to an increase in both spinach fructose-1,6-bisphosphatase and corn NADP-dependent malate dehydrogenase activities in the light-activation system. This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose-1,6-bisphosphatase and ferredoxin-dependent thioredoxin reductase.     

Thioredoxin system of the photosynthetic anaerobe Chromatium vinosum

Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. Both protein components were purified to homogeneity, and some of their properties were determined. Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties. As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m). The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.     

Thioredoxin and related proteins in procaryotes

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Oxidation-reduction properties of thioredoxins and thioredoxin-regulated enzymes

Oxidation-reduction midpoint potentials have been measured for the two chloroplast thioredoxins, thioredoxin f and m , for ferredoxin:thioredoxin reductase (FTR) and for the thioredoxin-regulated enzymes fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase and NADP-malate dehydrogenase. The effects of pH on the midpoint potentials of these chloroplast proteins have been measured so that the effect of the light-induced increase in chloroplast stromal pH on the redox properties of the proteins can be calculated. Spectroscopic measurements on FTR and on an N-ethylmaleimide-modified derivative of the enzyme have been used to elucidate the role of the [4Fe-4S] cluster of FTR during the reduction of the enzyme's active-site disulfide by ferredoxin.     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.     

Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction.

A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea. In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa). A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not. In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP-thioredoxin reductase (NTR) were evidenced. Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs. Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs. Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak. The algal thioredoxin h was also slightly active with spinach and E. coli NTRs. These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments.     

Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen.

The thioredoxin system, comprising NADPH, thioredoxin reductase and thioredoxin reduces protein disulfides via redox-active dithiols. We have discovered that sodium selenite is a substrate for the thioredoxin system; 10 microM selenite plus 0.05 microM calf thymus thioredoxin reductase at pH 7.5 caused a non-stoichiometric oxidation of NADPH (100 microM after 30 min). In contrast, thioredoxin reductase from Escherichia coli showed no direct reaction with selenite, but addition of 3 microM E. coli thioredoxin also resulted in non-stoichiometric oxidation of NADPH, consistent with oxidation of the two active-site thiol groups in thioredoxin to a disulfide. Kinetically, the reaction was complex with a lag phase at low selenite concentrations. Under anaerobic conditions the reaction stopped after 1 mol selenite had oxidized 3 mol NADPH; the admission of air then resulted in continued consumption of NADPH consistent with autooxidation of selenium intermediate(s). Ferricytochrome c was effectively reduced by calf thymus thioredoxin reductase and selenite in the presence of oxygen. Selenite caused a strong dose-dependent inhibition of the formation of thiol groups from insulin disulfides with either the E. coli or calf-thymus thioredoxin system. Thus, under aerobic conditions selenite catalyzed, NADPH-dependent redox cycling with oxygen, a large oxygen-dependent consumption of NADPH and oxidation of reduced thioredoxin inhibiting its disulfide-reductase activity.     

Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.     

The mechanism of photosynthetic sulfate reduction by isolated chloroplasts

The reduction of sulfate by isolated spinach chloroplasts was studied. A reconstituted system of broken chloroplasts and of chloroplast extract reduced sulfate to sulfite in the light when ADP, NADP⁺, ferredoxin and glutathione were added. The chloroplast extract reduced sulfate to sulfite in the dark if supplemented with ATP and with reduced glutathione. Neither ferredoxin nor NADPH were needed for this reduction in the dark.

A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP⁺ was not required for this reduction.

The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase.     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.     

Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system

A newly found form of chloroplast phosphoribulokinase (designated the “regulatory form”) required reduced thioredoxin for activity. A second form of the enzyme (the “nonregulatory form”) was not appreciably affected by thioredoxin. The thioredoxin required for activation of the regulatory enzyme could be reduced (i) photochemically by chloroplast membranes that were supplemented with ferredoxin and ferredoxin-thioredoxin reductase or (ii) chemically in the dark with the sulfhydryl reagent dithiothreitol. Following activation by reduced thioredoxin, phosphoribulokinase was deactivated by the soluble chloroplast oxidants dehydroascorbate and oxidized glutathione. The results suggest that the regulatory form of phosphoribulokinase resembles fructose 1,6-bisphosphatase in its mode of regulation by the ferredoxin/thioredoxin system.     

Thioredoxin reductase-mediated hydrogen transfer from Escherichia coli thioredoxin-(SH)2 to phage T4 thioredoxin-S2.

Thioredoxin from Escherichia coli B and phage T4-infected E. coli B are small hydrogen carrier proteins which in their reduced forms are specific hydrogen donors to E. coli and T4-induced ribonucleotide reductase, respectively. The oxidation-reduction active group of both thioredoxins consists of a single cystine residue which is reduced to sulfhydryl form by NADPH in the presence of E. coli thioredoxin reductase. Reduction of T4 thioredoxin-S2 to thioredoxin-(SH)2 led to a 3-fold increase in the quantum yield of tyrosine fluorescence. By using the spectrofluorimetric properties of T4 thioredoxin and E. coli thioredoxin as markers for their oxidized and reduced forms we have shown that E. coli thioredoxin reductase catalyzed the reaction: (see article) whose equilibrium constant favors formation of E. coli thioredoxin-S2 and T4 thioredoxin-(SH)2. This finding suggests that in the T4-infected cell most of the deoxyribonucleotides required for the viral DNA might be synthesized by the T4-induced ribonucleotide reductase while the host ribonucleotide reductase is inactive due to the shortage of reduced E. coli thioredoxin.     

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120.

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli. Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.     

Sulfate-reducing pathway in Escherichia coli involving bound intermediates.

Although a sulfate-reducing pathway in Escherichia coli involving free sulfite and sulfide has been suggested, it is shown that, as in Chlorella, a pathway involving bound intermediates is also present. E. coli extracts contained a sulfotransferase that transferred the sulfonyl group from a nucleosidephosphosulfate to an acceptor to form an organic thiosulfate. This enzyme was specific for adenosine 3'-phosphate 5'-phosphosulfate, did not utilize adenine 5'-phosphosulfate, and transferred to a carrier molecule that was identical with thioredoxin in molecular weight and amino acid composition. In the absence of thioredoxin, only very low levels of the transfer of the sulfo group to thiols was observed. As in Chlorella, thiosulfonate reductase activity that reduced glutathione-S-SO3- to bound sulfide could be detected. In E. coli, this enzyme used reduced nicotinamide adenine dinucleotide phosphate and Mg2+, but did not require the addition of ferredoxin or ferredoxin nicotinamide adenine dinucleotide phosphate reductase. Although in Chlorella the thiosulfonate reductase appears to be a different enzyme from the sulfite reductase, the E. coli thiosulfonate reductase and sulfite reductase may be activities of the same enzyme.     

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.