Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.

Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro and in Vivo Studies1

Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. GS in plants is an octameric enzyme. Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315–325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723–1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155–177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ·OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels.

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Trypanosoma brucei CTP synthetase: A target for the treatment of African sleeping sickness

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23–28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.

Comparative Analysis of the Regulation of Expression and Structures of Two Evolutionarily Divergent Genes for Δ1-Pyrroline-5-Carboxylate Synthetase from Tomato1

We isolated two tomato (Lycopersicon esculentum) cDNA clones, tomPRO1 and tomPRO2, specifying Δ1-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of proline (Pro) biosynthesis. tomPRO1 is unusual because it resembles prokaryotic polycistronic operons (M.G. García-Ríos, T. Fujita, P.C. LaRosa, R.D. Locy, J.M. Clithero, R.A. Bressan, L.N. Csonka [1997] Proc Natl Acad Sci USA 94: 8249–8254), whereas tomPRO2 encodes a full-length P5CS. We analyzed the accumulation of Pro and the tomPRO1 and tomPRO2 messages in response to NaCl stress and developmental signals. Treatment with 200 mm NaCl resulted in a >60-fold increase in Pro levels in roots and leaves. However, there was a <3-fold increase in the accumulation of the tomPRO2 message and no detectable induction in the level of the tomPRO1 message in response to NaCl stress. Although pollen contained approximately 100-fold higher levels of Pro than other plant tissues, there was no detectable increase in the level of either message in pollen. We conclude that transcriptional regulation of these genes for P5CS is probably not important for the osmotic or pollen-specific regulation of Pro synthesis in tomato. Using restriction fragment-length polymorphism mapping, we determined the locations of tomPRO1 and tomPRO2 loci in the tomato nuclear genome. Sequence comparison suggested that tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS genes.

Isolation and Characterization of Glutathione S-Transferase Isozymes from Sorghum1

Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4′-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapid-equilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I θ-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.

Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves1

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus1

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-14C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [14C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.

Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.