The conserved Cys76 plays a crucial role for the conformation of reduced glutathione peroxidase-type tryparedoxin peroxidase

The crystal structure of reduced tryparedoxin peroxidase shows Cys47 close to Gln82 and Trp137 and helix formation of residues 87 to 97 whereas the NMR structure of the reduced C76S mutant adopts a different conformation similar to the oxidized protein. Circular dichroism (CD), fluorescence and NMR spectroscopy reveal that the fully active C76S mutant differs from the wildtype (WT) enzyme mainly in its reduced form both in secondary structure content and Trp137 environment. This implies that Cys76 plays a critical role for the reduced enzyme assuming different conformational states and that the catalytic triad may only be necessary as short-lived intermediate during catalysis.     

Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61

Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys(95), Cys(82), and Cys(61), and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at Cys(61) generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys(61) as the peroxidatic center. In stopped-flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 x 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm). Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2). Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution.     

Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.     

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: A gene for alcohol-induced cardiomyopathy

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.     

Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH(2)-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only approximately 10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys(48)) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys(48) is the site of oxidation by peroxides and that oxidized Cys(48) reacts with the sulfhydryl group of Cys(152) to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys(48) or Cys(152) showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys(73) had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys(48) mutant, in NIH 3T3 cells inhibited H(2)O(2) accumulation and activation of c-Jun NH(2)-terminal kinase induced by tumor necrosis factor-alpha.     

Engineering out motion: a surface disulfide bond alters the mobility of tryptophan 22 in cytochrome b5 as probed by time-resolved fluorescence and 1H NMR experiments

In the accompanying paper [Storch et al. (1999) Biochemistry 38, 5054-5064] equilibrium denaturation studies and molecular dynamics (MD) simulations were used to investigate localized dynamics on the surface of cytochrome b5 (cyt b5) that result in the formation of a cleft. In those studies, an S18C:R47C disulfide mutant was engineered to inhibit cleft mobility. Temperature- and urea-induced denaturation studies revealed significant differences in Trp 22 fluorescence between the wild-type and mutant proteins. On the basis of the results, it was proposed that wild type populates a conformational ensemble that is unavailable to the disulfide mutant and is mediated by cleft mobility. As a result, the solvent accessibility of Trp 22 is decreased in S18C:R47C, suggesting that the local environment of this residue is less mobile due to the constraining effects of the disulfide on cleft dynamics. To further probe the structural effects on the local environment of Trp 22 caused by inhibition of cleft formation, we report here the results of steady-state and time-resolved fluorescence quenching, differential phase/modulation fluorescence anisotropy, and 1H NMR studies. In Trp fluorescence experiments, the Stern-Volmer quenching constant increases in wild type versus the oxidized disulfide mutant with increasing temperature. At 50 degrees C, KSV is nearly 1.5-fold greater in wild type compared to the oxidized disulfide mutant. In the reduced disulfide mutant, KSV was the same as wild type. The bimolecular collisional quenching constant, kq, for acrylamide quenching of Trp 22 increases 2.7-fold for wild type and only 1.8-fold for S18C:R47C, upon increasing the temperature from 25 to 50 degrees C. The time-resolved anisotropy decay at 25 degrees C was fit to a double-exponential decay for both the wild type and S18C:R47C. Both proteins exhibited a minor contribution from a low-amplitude fast decay, consistent with local motion of Trp 22. This component was more prevalent in the wild type, and the fractional contribution increased significantly upon raising the temperature. The fast rotational component of the S18C:R47C mutant was less sensitive to increasing temperature. A comparison of the 1H NMR monitored temperature titration of the delta-methyl protons of Ile 76 for wild type and oxidized disulfide mutant, S18C:R47C, showed a significantly smaller downfield shift for the mutant protein, suggesting that Trp 22 in the mutant protein experiences comparatively decreased cleft dynamics in core 2 at higher temperatures. Furthermore, comparison of the delta'-methyl protons of Leu 25 in the two proteins revealed a difference in the ratio of the equilibrium heme conformers of 1.2:1 for S18C:R47C versus 1.5:1 for wild type at 40 degrees C. The difference in equilibrium heme orientations between wild type and S18C:R47C suggests that the disulfide bond affects heme binding within core 1, possibly through damped cleft fluctuations. Taken together, the NMR and fluorescence studies support the proposal that an engineered disulfide bond inhibits the formation of a dynamic cleft on the surface of cyt b5.     

Identification and characterization of a critical region in the glycogen synthase from Escherichia coli

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.     

Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells

Glutamine (Gln) and keratinocyte growth factor (KGF) each stimulate intestinal epithelial cell growth, but regulatory mechanisms are not well understood. We determined whether Gln and KGF alter intra- and extracellular thiol/disulfide redox pools in Caco-2 cells cultured in oxidizing or reducing cell medium and whether such redox variations are a determinant of proliferative responses to these agents. Cells were cultured over a physiological range of oxidizing to reducing extracellular thiol/disulfide redox (Eh) conditions, obtained by varying cysteine (Cys) and cystine (CySS) concentrations in cell medium. Cell proliferation was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. Gln (10 mmol/l) or KGF (10 microg/l) did not alter BrdU incorporation at reducing Eh (-131 to -150 mV), but significantly increased incorporation at more oxidizing Eh (Gln at 0 to -109 mV; KGF at -46 to -80 mV). Cellular glutathione/glutathione disulfide (GSH/GSSG) Eh was unaffected by Gln, KGF, or variations in extracellular Cys/CySS Eh. Control cells largely maintained extracellular Eh at initial values after 24 h (-36 to -136 mV). However, extracellular Eh shifted toward a narrow physiological range with Gln and KGF treatment (Gln -56 to -88 mV and KGF -76 to -92 mV, respectively; P < 0.05 vs. control). The results indicate that thiol/disulfide redox state in the extracellular milieu is an important determinant of Caco-2 cell proliferation induced by Gln and KGF, that this control is independent of intracellular GSH redox status, and that both Gln and KGF enhance the capability of Caco-2 cells to modulate extremes of extracellular redox.     

Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.

Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.     

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Receptor binding activity and cytosolic free calcium response by synthetic endothelin analogs in cultured rat vascular smooth muscle cells

Using a variety of synthetic analogs of porcine endothelin (pET), we have studied the effects of these analogs on receptor binding activity and cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMC). Removal of C-terminal Trp21 residue, truncated derivatives pET(1-15) and (16-21), substitution of disulfide bond, Cys(3-11) or Cys(1-15), by Cys (Acm), all resulted in a complete loss of receptor binding activity and [Ca2+]i response, while N-terminal elongation of Lys-Arg residues, but not oxidation of Met7 residue, decreased receptor binding activity and [Ca2+]i response. [Cys1-15,Cys3-11]pET was far more potent than [Cys1-11,Cys3-15]pET in receptor binding and [Ca2+]i response. These data indicate that the C-terminal Trp21 as well as the proper double cyclic structure formed by the intramolecular disulfide bonds of the pET molecule are essential for receptor binding and subsequent [Ca2+]i increase in rat VSMC.     

Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.     

Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol-phospholipase C from Trypanosoma brucei.

Glycosylphosphatidylinositol-specific phospholipase C (GPtdIns-PLC) is found in the protozoan parasite Trypanosoma brucei. A region of protein sequence similarity exists between the protozoan enzyme and eubacterial phosphatidylinositol-phospholipases C. The functional relevance of Cys80 and Gln81 of GPtdIns-PLC, both in this region, was tested with a panel of mutations at each position. Gln81Glu, Gln81Ala, Gln81Gly, Gln81Lys and Gln81Leu mutants were inactive. Cleavage of GPtdIns was detectable in Gln81Asn, although the specific activity decreased 500-fold, and kcat was reduced 50-fold. Thus an amide side-chain at residue 81 is essential for catalysis by GPtdIns-PLC. Sulfhydryl reagents inactivate GPtdIns-PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly, p-chloromercuriphenyl sulfonate (p-CMPS) is significantly more potent than N-ethylmaleimide, the less bulky compound. This knowledge prompted us to test whether replacement of Cys80 with an amino acid possessing a bulky side-chain would inactivate GPtdIns-PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269-270-273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80Ala, Cys80Thr and Cys269-270-273Ser retained 33 to 100% of wild-type activity. Interestingly, the Cys80Ala and Cys80Thr mutants became resistant to p-CMPS, as predicted if the sulfhydryl reagent reacted with Cys80 in the wild-type enzyme to form a cysteinyl mercurylphenylsulfonate moiety, a bulky adduct that inactivated GPtdIns-PLC, similar to the Cys80Phe mutation. We conclude that a bulky side-chain (or adduct) at position 80 of GPtdIns-PLC abolishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns-PLC from T. brucei.     

Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite

Thioredoxin peroxidase 1 (TPx1) of the malarial parasite Plasmodium falciparum is a 2-Cys peroxiredoxin involved in the detoxification of reactive oxygen species and - as shown here - of reactive nitrogen species. As novel electron acceptor of reduced TPx1, we characterised peroxynitrite; the rate constant for ONOO- reduction by the enzyme (1 x 10(6) M(-1) s(-1) at pH 7.4 and 37 degrees C) was determined by stopped-flow measurements. As reducing substrate of TPx1, we identified - aside from thioredoxin - plasmoredoxin; this 22-kDa protein occurs only in malarial parasites. When studying the potential roles of Cys74 and Cys170 of Tpx1 in catalysis, as well as in oligomerisation behaviour, we found that replacement of Cys74 by Ala influenced neither the dimerisation nor enzymatic activity of TPx1. In the C170A mutant, however, the kcat/Km for reduced Trx as a substrate was shown to be approximately 50-fold lower and, in contrast to the wild-type enzyme, covalently linked dimers were not formed. For the catalytic cycle of TPx1, we conclude that oxidation of the peroxidatic Cys50 by the oxidising substrate is followed by the formation of an intermolecular disulfide bond between Cys50 and Cys170' of the second subunit, which is then attacked by an external electron donor such as thioredoxin or plasmoredoxin.     

Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106)

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.     

Mutational hotspots of HSP47 and its potential role in cancer and bone-disorders

Heat shock protein 47 kDa (HSP47) serves as a client-specific chaperone, essential for collagen biosynthesis and its folding and structural assembly. To date, there is no comprehensive study on mutational hotspots. Using five different human mutational databases, we deduced a comprehensive list of human HSP47 mutations with 24, 67, 50, 43 and 2 deleterious mutations from the 1000 genomes data, gnomAD, COSMICv86, cBioPortal, and CanVar, respectively. We identified thirteen top-ranked missense mutations of HSP47 with the stringent cut-off of CADD score (>25) and Grantham score (≥151) as Ser76Trp, Arg103Cys, Arg116Cys, Ser159Phe, Arg167Cys, Arg280Cys, Trp293Cys, Gly323Trp, Arg339Cys, Arg373Cys, Arg377Cys, Ser399Phe, and Arg405Cys with the arginine-cysteine changes as the predominant mutations. These findings will assist in the evaluation of roles of HSP47 in collagen misfolding and human diseases such as cancer and bone disorders.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.     

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.