Cloning and characterization of glyoxalase I from soybean

Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No. X68819) resembling a GST being described as a glyoxalase I. To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures. When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or gamma-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean. When the clone X68819 was expressed in E. coli, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3. GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzymes are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%).     

Control of cell proliferation and differentiation by regulating polyamine biosynthesis in cultures of Brassica and its correlation with glyoxalase-I activity

Differentiation in Brussica cultures could be induced on basal medium lacking hormones, while addition of hormones (NAA, BA) resulted in profuse callusing without any differentiation. Supplementing the hormone medium with spermidine resulted in increase in the fresh weight and glyoxalase-I activity by 330% and 8-fold, respectively. Omission of hormones caused spermidine to be less effective in inducing either cell proliferation or differentiation. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, had a retarding effect on callus induction and division of cells in suspension cultures but lead to differentiation and inhibited glyoxalase-I activity. The ability of spermidine to overcome MGBG enhanced differentiation was probably through the breaking of cell cycle arrest. Addition of glutathione, a coenzyme for glyoxalase-I enzyme, promoted cell division and enzyme activity both in callus and suspension cultures. pH emerged as an important factor in controlling glyoxalase-I activity and cell division. Results indicate involvement of spermidine in cell proliferation and differentiation and its correlation with glyoxalase-I activity.     

Rearrangement of enzyme patterns in maize callus and suspension cultures

The development of enzyme patterns was followed in the course of: (a) the irreversible cell differentiation via division and expansion to maturity in the root tip and coleoptile of the intact seedlings, (b) the irreversible cell dedifferentation associated with induction and establishment of callus from the growing internodes, and (c) the growth cycle (proliferationstationary phase) in callus and cell-suspension cultures of maize (Zea mays L.). By measuring the activities of glycolytic, mitochondrial, microbody and hydrolytic enzymes cells proliferating in vivo and in vitro could be compared and changes related to cessation or resumption of cell division could be studied.Proliferating cells of callus and suspension cultures maintained by serial culture did not differ from those of the root meristem and coleoptile in the specific activities of hexokinase, phosphoglycerate kinase and phosphopyruvate hydratase. Proliferation in vitro resulted in an enormous increase in the ratio g glutamate-dehydrogenase/cytochrome-oxidase activity and in the level of acid-phosphatase activity, with concomitant drop in galactosidase and xylosidase activity. A 3-5-fold increase of alcohol-dehydrogenase, lactate-dehydrogenase and catalase activities was characteristic of dividing callus cells, while a ca. 100-fold increase in the fructofuranosidase-to-glucosidase activity ratio marked cell proliferation in suspension-cultured cells.Changing enzyme activities after cessation of proliferation were quite similar in root tips and coleoptiles, except those of alcohol dehydrogenase and catalase. The enzyme rearrangement during callus establishment and in the growth cycle of callus cultures was in most cases comparable to that in the intact tissues, while the changes from the dividing to the non-dividing cells in suspension cultures, in contrast, differed widely from those in the intact tissues and callus. Galactosidase and xylosidase were the only activities that showed a similar trend of changes in all the investigated, intact and in-vitro-grown cells.Thus, judged by the pattern of enzyme development, the cell suspension appears to be a unique system, virtually unrelated to the growing cells of the intact tissues. It is also very difficult to draw a definite distinction between the metabolic consequences of cell growth and enzyme modulations in cell suspensions as the cells adapt their metabolism to the environmental changes in liquid medium.     

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.     

The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli

The glyoxalase I gene ( gloA ) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E . coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.     

Activation of glyoxalase I during the cell division cycle and its homology with auxin regulated genes

In several plant systems increase in glyoxalase I activity has been correlated with cell proliferation. Cell cycle studies of tobacco protoplasts indicate a rise in glyoxalase I activity prior to G₂/M phase. Further, synthetic auxin, NAA, induced glyoxalase I activity and cell division significantly. This induction was specific in response to auxin only. Cytokinins alone do not induce cell division or increase enzyme activity. Analysis of glyoxalase I cDNA sequence from soybean shows significant homology with auxin inducible genes particularly Nt107 and limited but strong similarity with identified plant mitotic cyclins, implicating glyoxalase I in possible relationship with certain cell division regulating factors.     

Proline and glycinebetaine enhance antioxidant defense and methylglyoxal detoxification systems and reduce NaCl-induced damage in cultured tobacco cells

Salt stress impairs reactive oxygen species (ROS) and methylglyoxal (MG) detoxification systems, and causes oxidative damage to plants. Up-regulation of the antioxidant and glyoxalase systems provides protection against NaCl-induced oxidative damage in plants. Thiol–disulfide contents, glutathione content and its associated enzyme activities involved in the antioxidant defense and glyoxalase systems, and protein carbonylation in tobacco Bright Yellow-2 cells grown in suspension culture were investigated to assess the protection offered by proline and glycinebetaine against salt stress. Salt stress increased protein carbonylation, contents of thiol, disulfide, reduced (GSH) and oxidized (GSSG) forms of glutathione, and the activity of glutathione-S-transferase and glyoxalase II enzymes, but decreased redox state of both thiol–disulfide and glutathione, and the activity of glutathione peroxidase and glyoxalase I enzymes involved in the ROS and MG detoxification systems. Exogenous application of proline or glycinebetaine resulted in a reduction of protein carbonylation, and in an increase in glutathione redox state and activity of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. Neither proline nor glycinebetaine, however, had any direct protective effect on NaCl-induced GSH-associated enzyme activities. The present study, therefore, suggests that both proline and glycinebetaine provide a protective action against NaCl-induced oxidative damage by reducing protein carbonylation, and enhancing antioxidant defense and MG detoxification systems.     

Correlation of glyoxalase I activity with cell proliferation inDatura callus culture

Glyoxalase-I activity in growingDatura callus showed 184% increase with the age of the culture. Spermidine increased the enzyme activity together with DNA and protein synthesis. With the addition of mitotic inhibitors, vinblastine and methylglyoxal in the growth medium, the enzyme activity was inhibited by 92 percent and 50 percent respectively, at the most effective concentration and the callus growth was also reduced. Similar results were obtained with specific glyoxalase I inhibitors, iso-ascorbate and squaric acid.     

Role of the N-terminus of glutathione in the action of yeast glyoxalase I.

A number of S-substituted glutathiones and the corresponding N-substituted S-substituted analogues have been found to be linear competitive inhibitors of yeast glyoxalase I at 26 degrees C over the pH range 4.6-8.5. N-Acetylation of S-(p-bromobenzyl)glutathione weakens binding by 13.7-fold. N-benzoylation by 25.6-fold, N-trimethylacetylation by 53.3-fold and N-carbobenzoxylation by 7.8-fold, indicating a minor steric component in the binding at the N-site. The Ki-weakening effect of N-substitution of glutathione depends on the chemical nature of the S-substituent, indicating flexibility in the glutathione and/or glyoxalase I contributions to the binding site for glutathione derivatives. The effect of N-acylation on Ki is in accord with a charge interaction of the free enzyme with S-blocked glutathione in a region of reasonably high dielectric constant. There is a slight pH effect on Ki for S-(m-trifluoromethylbenzyl)glutathione but not for S-(p-bromobenzyl)glutathione.     

Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc-spiked soils

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.     

Inhibition of glyoxalase I by the enediol mimic S-(N-hydroxy-N-methylcarbamoyl)glutathione. The possible basis of a tumor-selective anticancer strategy.

In principle, competitive inhibitors of glyoxalase I that also serve as substrates for the thioester hydrolase glyoxalase II might function as tumor-selective anti-cancer agents, given the role of these enzymes in removing cytotoxic methylglyoxal from cells and the observation that glyoxalase II activity is abnormally low in some types of cancer cells. In support of the feasibility of this anticancer strategy, an inhibitor of this type has been synthesized by a thioester-interchange reaction between glutathione and N-hydroxy-N-methylcarbamate 4-chlorophenyl ester to give S-(N-hydroxy-N-methylcarbamoyl)glutathione (1). This compound was designed to be a tight-binding inhibitor of glyoxalase I, on the basis of its stereoelectronic similarity to the enediol(ate) intermediate that forms along the reaction pathway of this enzyme. Indeed, 1 is a competitive inhibitor of yeast glyoxalase I, with an inhibition constant (Ki = 68 microM) that is approximately 30-fold lower than that reported for S-D-lactoylglutathione and approximately 7-fold lower than the Km for glutathione-methylglyoxal thiohemiacetal. In addition, 1 is a substrate for bovine liver glyoxalase II, with a Km (0.48 mM) approximately equal to that of the normal substrate S-D-lactoyglutathione and a kcat approximately 2 x 10(-5)-fold that of the normal substrate. Membrane transport studies show that 1 can be delivered into human erythrocytes (used here as a model cell) either by direct diffusion of 1 across the cell membrane or by more rapid diffusion of the glycylethyl ester of 1 across the cell membrane, followed by the catalyzed hydrolysis of the ester to give 1.     

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.     

Physiological and biochemical characterization of glyoxalase I,a general marker for cell proliferation,from a soybean cell suspension

Using a strictly auxin-dependent soybean (Glycine max (L.) Merr.) cell suspension, we studied the correlation of auxin-dependent cell proliferation and the activity of glyoxalase I (S-lactoylglutathione-lyase EC 4.4.1.5.), an enzyme generally associated with cell proliferation in animal, microbial and, as reported recently, also plant systems. We found the activity of glyoxalase I to be modulated during the proliferation cycle, with a maximal activity between day 2 and day 4 of culture growth. After starving the culture of auxins for three subsequent periods, both the enzyme activity and cell growth could be re-initiated with auxin. Enzyme activity reached its maximum 1 d before cell number was at a maximum. The enzyme was purified to homogeneity and characterized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GSH reuced glutathione - Mr relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors thank Dr. K. Palme, Max-Planck-Institute, Cologne, for reverse-phase chromatography. Part of this work was done by C. Paulus at the Department of Biotechnology, New Delhi, India under the Bundesministerium für Forschung und Technologie (BMFT)-funded Indo-FRG collaboration programme. Thanks are due to Professors S. Guha-Mukherjee and S.K. Sopory, New Delhi, for introduction into glyoxalase research. The research was funded by a BMFT-DECHEMA fellowship to C. Paulus, a BMFT grant to H.-J. Jacobsen and a Graduierten Förderung des Landes Nordrhein-Westfalen fellowship to B. Köllner.     

Selenium Pretreatment Upregulates the Antioxidant Defense and Methylglyoxal Detoxification System and Confers Enhanced Tolerance to Drought Stress in Rapeseed Seedlings

In order to observe the possible regulatory role of selenium (Se) in relation to the changes in ascorbate (AsA) glutathione (GSH) levels and to the activities of antioxidant and glyoxalase pathway enzymes, rapeseed (Brassica napus) seedlings were grown in Petri dishes. A set of 10-day-old seedlings was pretreated with 25 μM Se (Sodium selenate) for 48 h. Two levels of drought stress (10% and 20% PEG) were imposed separately as well as on Se-pretreated seedlings, which were grown for another 48 h. Drought stress, at any level, caused a significant increase in GSH and glutathione disulfide (GSSG) content; however, the AsA content increased only under mild stress. The activity of ascorbate peroxidase (APX) was not affected by drought stress. The monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activity increased only under mild stress (10% PEG). The activity of dehydroascorbate reductase (DHAR), glutathione S-transferase (GST), glutathione peroxidase (GPX), and glyoxalase I (Gly I) activity significantly increased under any level of drought stress, while catalase (CAT) and glyoxalase II (Gly II) activity decreased. A sharp increase in hydrogen peroxide (H₂O₂) and lipid peroxidation (MDA content) was induced by drought stress. On the other hand, Se-pretreated seedlings exposed to drought stress showed a rise in AsA and GSH content, maintained a high GSH/GSSG ratio, and evidenced increased activities of APX, DHAR, MDHAR, GR, GST, GPX, CAT, Gly I, and Gly II as compared with the drought-stressed plants without Se. These seedlings showed a concomitant decrease in GSSG content, H₂O₂, and the level of lipid peroxidation. The results indicate that the exogenous application of Se increased the tolerance of the plants to drought-induced oxidative damage by enhancing their antioxidant defense and methylglyoxal detoxification systems.     

Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene.

Anaerobic glycerol fermentation by Escherichia coli strains expressing genes from the Klebsiella pneumoniae dha regulon showed that cell growth and 1,3-propanediol (1,3-PD) production are significantly inhibited when 5 g/L or higher of glycerol is initially present. One reason for this inhibition may be methylglyoxal (MG) accumulation. Assays of both intracellular and extracellular MG levels indicated an accumulation of MG in anaerobic glycerol fermentation of transgenic E. coli. Pseudomonas putida glyoxalase I was expressed in the transgenic E. coli to enhance MG detoxification. The activity of glyoxalase I in the transgenic E. coli with the P. putida glyoxalase I under anaerobic conditions was 12-fold higher than that in the control cells. Compared to the control cells, the transgenic cells with the P. putida glyoxalase I displayed a reduction of 35-43% in intracellular MG and a decrease of 30% in extracellular MG. These decreases were statistically significant (P>94). Furthermore, the expression of the P. putida glyoxalase I in the transgenic E. coli markedly improved cell growth and resulted in a 50% increase in 1,3-PD production.     

Methylglyoxal as a novel signal molecule induces the salt tolerance of wheat by regulating the glyoxalase system,the antioxidant system,and osmolytes

Reactive carbonyl species methylglyoxal (MG) has always been regarded as a cytotoxic metabolite, but now is emerging to function as signal molecule in plants. However, whether MG can induce salt tolerance is elusive. In this study, treatment of wheat seeds with NaCl reduced seed germination, plant height, root length, fresh weight, and dry weight, indicating the inhibitive effects of NaCl on seed germination and seedling growth. The inhibitive effects of NaCl were alleviated by applying exogenous MG, but aggravated by the MG scavenger N-acetyl-L-cysteine (NAC), suggesting that MG could induce the salt tolerance of wheat. In addition, MG increased glyoxalase I and glyoxalase II activities and decreased endogenous MG content in wheat seedlings under NaCl stress, whereas coapplication of NAC weakened glyoxalase activity and enhanced the endogenous MG level. Also, MG activated superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase activities; increased glutathione and ascorbic acid levels; and decreased superoxide radical production and H₂O₂ and malondialdehyde contents under NaCl stress, while NAC reversed these physiological parameters. Furthermore, MG also induced the accumulation of proline, glycine betaine, and soluble sugar under NaCl stress, whereas this accumulation was weakened by NAC. This work reported for the first time that MG could induce the salt tolerance of wheat, and the acquisition of this salt tolerance was involved in the activation of the glyoxalase system and antioxidant system, as well as the accumulation of osmolytes.     

Ethephon mitigates nickel stress by modulating antioxidant system,glyoxalase system and proline metabolism in Indian mustard

The role of ethylene (through application of ethephon) in the regulation of nickel (Ni) stress tolerance was investigated in this study. Ethephon at concentration of 200 µl l^?1 was applied to mustard (Brassica juncea) plants grown without and with 200 mg kg^?1 soil Ni to study the increased growth traits, biochemical attributes, photosynthetic efficiency, nutrients content, activities of antioxidants such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase, glyoxalase systems and enhanced the proline metabolism. In the absence of ethephon, Ni increased oxidative stress with a concomitant decrease in photosynthesis, growth and nutrients content. However, application of ethephon positively increased growth traits, photosynthetic parameters, nutrients content and also elevated the generation of antioxidants enzymes and glyoxalase systems, proline production to combat oxidative stress. Plants water relations and cellular homeostasis were maintained through increased photosynthetic efficiency and proline production. This signifies the role of ethylene in mediating Ni tolerance via regulating proline production and photosynthetic capacity. Ethephon can be used as an exogenous supplement on plants to confer Ni tolerance. The results can be exploited to develop tolerance in plants via gene editing technology encoding enzymes responsible for proline synthesis, antioxidant defence, glyoxalase systems and photosynthetic effectiveness.

     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.