NrdH-redoxin of Corynebacterium ammoniagenes forms a domain-swapped dimer

NrdH-redoxins constitute a family of small redox proteins, which contain a conserved CXXC sequence motif, and are characterized by a glutaredoxin-like amino acid sequence but a thioredoxin-like activity profile. Here we report the structure of Corynebacterium ammoniagenes NrdH at 2.7 A resolution, determined by molecular replacement using E. coli NrdH as model. The structure is the first example of a domain-swapped dimer from the thioredoxin family. The domain-swapped structure is formed by an inter-chain two-stranded anti-parallel beta-sheet and is stabilized by electrostatic interactions at the dimer interface. Size exclusion chromatography, and MALDI-ESI experiments revealed however, that the protein exists as a monomer in solution. Similar to E. coli NrdH-redoxin and thioredoxin, C. ammoniagenes NrdH-redoxin has a wide hydrophobic pocket at the surface that could be involved in binding to thioredoxin reductase. However, the loop between alpha2 and beta3, which is complementary to a crevice in the reductase in the thioredoxin-thioredoxin reductase complex, is the hinge for formation of the swapped dimer in C. ammoniagenes NrdH-redoxin. C. ammoniagenes NrdH-redoxin has the highly conserved sequence motif W61-S-G-F-R-P-[DE]67 which is unique to the NrdH-redoxins and which determines the orientation of helix alpha3. An extended hydrogen-bond network, similar to that in E. coli NrdH-redoxin, determines the conformation of the loop formed by the conserved motif.     

A thioredoxin from the hyperthermophilic archaeon Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities

A thioredoxin homologue (Mj0307) from the hyperthermophilic archaeon Methanococcus jannaschii (MjTRX) was cloned, produced in E. coli, and compared to the thioredoxin from E. coli (ETRX). The secondary structure profile of MjTRX obtained by NMR spectroscopy shows that it has four beta-sheets and three alpha-helices arranged in betaalphabetaalphabetabetaalpha, similar to that of glutaredoxin. However, MjTRX supports the growth of T7 bacteriophage in E. coli and is weakly reduced by the thioredoxin reductase from E. coli, indicating that MjTRX is functionally closer to a thioredoxin than a glutaredoxin. MjTRX has higher specific insulin reductase activity than ETRX and retained its full activity over 4 days at 95 degrees C, whereas ETRX lost its activity in 150 min. The standard state redox potential of MjTRX is about -277 mV, which is the lowest value thus far known among redox potentials of the thioredoxin superfamily. This indicates that the lower redox potential is necessary in keeping catalytic disulfide bonds reduced in the cytoplasm and in coping with oxidative stress in an anaerobic hyperthermophile.     

A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii

A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii. These two genes were cloned and the corresponding expressed proteins were characterized. The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity. The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178). The protein from PH1426 was a typical, homodimeric flavoprotein. These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P. horikoshii. The redox protein and thioredoxin reductase retained their full activities for over 1h at 100 degrees C. The redox potential of the redox protein was similar to that of thioredoxin from E. coli and lower than that of glutathione. Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence.     

Thioredoxin/Glutaredoxin System of Chlorella: CHLORELLA ADENOSINE 5'-PHOSPHOSULFATE SULFOTRANSFERASE CANNOT USE THIOREDOXIN OR GLUTAREDOXIN AS COFACTORS

Using the thioredoxin/glutaredoxin-dependent adenosine 3'-phosphate 5'-phosphosulfate reductase coupled assay system, the Chlorella thioredoxin/glutaredoxin system has been partially purified and characterized. A NADPH-thioredoxin reductase and two thioredoxin/glutaredoxin activities, designated as Chlorella thioredoxin/glutaredoxin protein I and II (CPI and CPII), were found in crude extracts of Chlorella. Similar to their counterparts from Escherichia coli, both CPI and CPII are heat-stable low molecular proteins of approximately 14,000. While CPI (but not CPII) is a substrate for its homologous NADPH-thioredoxin reductase as well as for E. coli NADPH-thioredoxin reductase, CPII is better than CPI as a substrate for reduction by the glutathione system. Based on these properties, CPI and CPII may be classified as Chlorella thioredoxin and Chlorella glutaredoxin, respectively. The Chlorella NADPH-thioredoxin reductase (M(r) = 72,000, with two 36,000-dalton subunits) resembles E. coli-thioredoxin reductase in size. Besides Chlorella thioredoxin, the Chlorella thioredoxin reductase will also use E. coli thioredoxin, but not glutaredoxin, as a substrate. Although a thioredoxin-like protein has been implicated in higher plant light-dependent sulfate reaction, neither Chlorella thioredoxin nor glutaredoxin can stimulate the thiol-dependent adenosine 5'-phosphosulfate-sulfotransferase reaction. Furthermore, Chlorella thioredoxin and glutaredoxin, in conjunction with an appropriate reductase system, cannot replace the thiol requirement of Chlorella adenosine 5'-phosphosulfate-sulfotransferase. The exact physiological roles and subcellular localization of the Chlorella thioredoxin and glutaredoxin systems remain to be determined.     

Assimilatory sulfate reduction in Escherichia coli: identification of the alternate cofactor for adenosine 3'-phosphate 5'-phosphosulfate reductase as glutaredoxin.

The alternate cofactor (7004 cofactor) for Escherichia coli adenosine 3'-phosphate 5'-phosphosulfate (PAPS) reductase originally discovered in an E. coli mutant (tsnC 7004) lacking thioredoxin activity has now been purified and characterized. The tryptic peptide map of the 7004 cofactor is totally different from that of thioredoxin, indicating that the two proteins are unrelated in their primary structure. The 7004 cofactor has an amino acid composition different from that of thioredoxin but similar to that of glutaredoxin, a protein required for the glutathione-dependent deoxyribonucleotide formation by ribonucleotide reductase. Thus, the 7004 cofactor could not be a mutated form of thioredoxin, as was suspected earlier. Thioredoxin but not glutaredoxin is a substrate for thioredoxin reductase, but both thioredoxin and glutaredoxin can catalyze the dithiothreitol- or glutathione-dependent reduction of PAPS. On a molar basis, the dithiothreitol-coupled cofactor activity of thioredoxin is three- to fourfold higher that that of glutaredoxin. Comparison of the cofactor activities in the glutathione-coupled and the dithiothreitol-coupled PAPS reductase reaction shows that the cofactor activity of thioredoxin in the glutathione-coupled reaction is only 23% of that observed in the dithiothreitol-coupled reaction. However, in the case of glutaredoxin, cofactor activities are approximately the same in both the dithiothreitol- and glutathione-coupled reactions.     

Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).     

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.     

Plasmoredoxin, a novel redox-active protein unique for malarial parasites.

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development. Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m-1.s-1 at pH 6.9 and 25 degrees C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites. Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.     

NMR structure of oxidized Escherichia coli glutaredoxin: comparison with reduced E. coli glutaredoxin and functionally related proteins.

The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet. The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional [15N,1H]-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent-accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.     

Modifications of the active center of T4 thioredoxin by site-directed mutagenesis

The active site sequence of T4 thioredoxin, Cys-Val-Tyr-Cys, has been modified in two positions to Cys-Gly-Pro-Cys to mimic that of Escherichia coli thioredoxin. The two point mutants Cys-Gly-Tyr-Cys and Cys-Val-Pro-Cys have also been constructed. The mutant proteins have similar reaction rates with T4 ribonucleotide reductase as has the wild-type T4 thioredoxin. Mutant T4 thioredoxins with Pro instead of Tyr at position 16 in the active site sequence have three to four times lower apparent KM with E. coli ribonucleotide reductase than wild-type T4 thioredoxin. The KM values for these mutant proteins which do not have Tyr in position 16 are thus closer to E. coli thioredoxin than to the wild-type T4 thioredoxin. The bulky tyrosine side chain probably prevents proper interactions to E. coli ribonucleotide reductase. Also the redox potentials of these two mutant thioredoxins are lower than that of the wild-type T4 thioredoxin and are thereby more similar to the redox potential of E. coli thioredoxin. Mutations in position 15 behave more or less like the wild-type protein. The kinetic parameters with E. coli thioredoxin reductase are similar for wild-type and mutant T4 thioredoxins except that the apparent kcat is lower for the mutant protein with Pro instead of Tyr in position 16. The active site sequence of T4 thioredoxin has also been changed to Cys-Pro-Tyr-Cys to mimic that of glutaredoxins. This change does not markedly alter the reaction rate of the mutant protein with T4 ribonucleotide reductase or E. coli thioredoxin reductase, but the redox potential is lower for this mutant protein than for wild-type T4 thioredoxin.     

Sequence of thioredoxin reductase from Escherichia coli. Relationship to other flavoprotein disulfide oxidoreductases

The DNA sequence of the Escherichia coli gene encoding thioredoxin reductase has been determined. The predicted protein sequence agrees with an earlier determination of the 17 amino-terminal amino acids and with a fragment of the protein containing the redox-active half-cystines. Similarity between E. coli thioredoxin reductase and other flavoprotein disulfide oxidoreductases is quite limited, but three short segments, two of which are probably involved in FAD and NADPH binding, are highly conserved between thioredoxin reductase, glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase.     

Thioredoxin and related proteins in procaryotes

Abstract Thioredoxin is a small ( M _r 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 ferrodoxin; 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.     

Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.     

NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum Dimerizes at High Protein Concentration and Exclusively Receives Electrons from Thioredoxin Reductase

NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10⁶ and 5.6 × 10⁶ m⁻¹ min⁻¹, respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pK_a lower than 6.5 for nucleophilic attack, whereas the pK_a of the C-terminal cysteine is ∼10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum.     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.     

Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.     

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.     

3'-Phosphoadenosine-5'-phosphosulfate reductase in complex with thioredoxin: a structural snapshot in the catalytic cycle

The crystal structure of Escherichia coli 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase in complex with E. coli thioredoxin 1 (Trx1) has been determined to 3.0 A resolution. The two proteins are covalently linked via a mixed disulfide that forms during nucleophilic attack of Trx's N-terminal cysteine on the Sgamma atom of the PAPS reductase S-sulfocysteine (E-Cys-Sgamma-SO3-), a central intermediate in the catalytic cycle. For the first time in a crystal structure, residues 235-244 in the PAPS reductase C-terminus are observed, depicting an array of interprotein salt bridges between Trx and the strictly conserved glutathione-like sequence, Glu238Cys239Gly240Leu241His242. The structure also reveals a Trx-binding surface adjacent to the active site cleft and regions of PAPS reductase associated with conformational change. Interaction at this site strategically positions Trx to bind the S-sulfated C-terminus and addresses the mechanism for requisite structural rearrangement of this domain. An apparent sulfite-binding pocket at the protein-protein interface explicitly orients the S-sulfocysteine Sgamma atom for nucleophilic attack in a subsequent step. Taken together, the structure of PAPS reductase in complex with Trx highlights the large structural rearrangement required to accomplish sulfonucleotide reduction and suggests a role for Trx in catalysis beyond the paradigm of disulfide reduction.