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.     

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.     

A reinvestigation of inhibitors of glyoxalase I

It has been reported earlier that nucleotides, nucleosides and a series of structurally related compounds as well as compounds based on transition state analogy inhibit yeast glyoxalase I. In our study on the metabolic regulation of glyoxalase I, we have found that nucleotides such as ATP, GTP and different classes of other reagents based on transition state analogy (D-isoascorbate, dihydroxyfumaric acid, rhodizonic acid) do not inhibit yeast or goat liver glyoxalase I. The reported inhibition of glyoxalase I by these compounds has been found to be due to the interference of these compounds with the absorbancy at 240 nm of S-D-lactoylglutathione formed by the glyoxalase I reaction. Glyoxalase I from goat liver has been found to be strongly and competitively inhibited by lactaldehyde. But, lactaldehyde has very little inhibitory effect on yeast glyoxalase I. Lactaldehyde is formed from methylglyoxal, the substrate for glyoxalase I by the enzyme methylglyoxal reductase. D-Lactaldehyde inhibits the liver enzyme more strongly than L-lactaldehyde.     

Overexpression of Glyoxalase III gene in transgenic sugarcane confers enhanced performance under salinity stress

Journal of Plant Research - The glyoxalase pathway is a check point to monitor the elevation of methylglyoxal (MG) level in plants and is mediated by glyoxalase I (Gly I) and glyoxalase II (Gly II)...     

Catalysis and structural properties of Leishmania infantum glyoxalase II: trypanothione specificity and phylogeny

The glyoxalase pathway catalyzes the formation of d-lactate from methylglyoxal, a toxic byproduct of glycolysis. In trypanosomatids, trypanothione replaces glutathione in this pathway, making it a potential drug target, since its selective inhibition might increase methylglyoxal concentration in the parasites. Two glyoxalase II structures were solved. One with a bound spermidine molecule (1.8 A) and the other with d-lactate at the active site (1.9 A). The second structure was obtained by crystal soaking with the enzyme substrate (S)-d-lactoyltrypanothione. The overall structure of Leishmania infantum glyoxalase II is very similar to its human counterpart, with important differences at the substrate binding site. The crystal structure of L. infantum glyoxalase II is the first structure of this enzyme from trypanosomatids. The differential specificity of glyoxalase II toward glutathione and trypanothione moieties was revealed by differential substrate binding. Evolutionary analysis shows that trypanosomatid glyoxalases II diverged early from eukaryotic enzymes, being unrelated to prokaryotic proteins.     

Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme

Cytotoxic methylglyoxal is detoxified by the two-enzyme glyoxalase system. Glyoxalase I (GlxI) catalyzes conversion of non-enzymatically produced methylglyoxal-glutathione hemithioacetal into its corresponding thioester. Glyoxalase II (Glx II) hydrolyzes the thioester into d-lactate and free glutathione. Glyoxalase I and II are metalloenzymes, which possess mononuclear and binuclear active sites, respectively. There are two distinct classes of GlxI; the first class is Zn2+-dependent and is composed of GlxI from mainly eukaryotic organisms and the second class is composed of non-Zn2+-dependent (but Ni2+ or Co2+-dependent) GlxI enzymes (mainly prokaryotic and leishmanial species). GlxII is typically Zn2+-activated, containing Zn2+ and either Fe3+/Fe2+ or Mn2+ at the active site depending upon the biological source. To address whether two classes of GlxII might exist, glyoxalase II from Escherichia coli was cloned and overexpressed and characterized. Unlike E. coli GlxI, which is non-Zn2+-dependent, Zn2+ activates the E. coli GlxII enzyme, with no evidence for Ni2+ metal utilization.     

Modification of the glyoxalase system in human HL60 promyelocytic leukaemia cells during differentiation to neutrophils in vitro

The glyoxalase system of human promyelocytic leukaemia HL60 cells was substantially modified during differentiation to neutrophils. The activity of glyoxalase I was decreased and the activity of glyoxalase II was markedly increased relative to the level in control HL60 promyelocytes. There was a decrease in the apparent maximum velocity, Vmax, of glyoxalase I, and an increase in the Vmax of glyoxalase II. The apparent Michaelis constants for both enzymes remained unchanged. The flux of intermediates metabolised via the glyoxalase system increased during differentiation, as judged by the formation of D-lactic acid, whereas the percentage of glucotriose metabolised via the glyoxalase system remained unchanged. The cellular concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, were markedly decreased during differentiation. The maturation of HL60 promyelocytes is associated with an increased ability to metabolise S-D-lactoylglutathione by glyoxalase II and a concomitant decrease in the mean intracellular concentrations of S-D-lactoylglutathione and methylglyoxal. The maintenance of a high concentration of S-D-lactoylglutathione in HL60 promyelocytes may be related to the status of the microtubular cytoskeleton, since S-D-lactoylglutathione potentiates the GTP-promoted assembly of microtubules.     

The glutathione-dependent glyoxalase pathway in the yeast Saccharomyces cerevisiae

Glyoxalase I (EC 4.4.1.5), which catalyzes the reaction methylglyoxal + GSH leads to S-lactoylglutathione, is a ubiquitous enzyme for which no clear physiological function has been shown. In the yeast Saccharomyces cerevisiae, methylglyoxal may derive from the spontaneous decay of intracellular glyceraldehyde-3-P, which may accumulate during growth on glycerol as the carbon source. The half-life time for the triose phosphate was found to be 4.6 h under physiological conditions (pH 6.2, 0.05 M phosphate at 30 degrees C). Glyoxalase I is induced by growth on glycerol or by the addition of methylglyoxal to the growth medium. The enzyme is also subject to carbon catabolite repression. A mutant strain, fully defective in glyoxalase I and bearing only one nuclear mutation, was obtained. The strain, which is killed by exposure to glycerol, excretes methylglyoxal into the medium. Growth of the mutant on glucose as carbon source appears to be similar to that of the wild type strain. This investigation has clearly demonstrated a physiological role of glyoxalase I in a eucaryotic cell.     

Optimization of efficiency in the glyoxalase pathway

A quantitative kinetic model for the glutathione-dependent conversion of methylglyoxal to D-lactate in mammalian erythrocytes has been formulated, on the basis of the measured or calculated rate and equilibrium constants associated with (a) the hydration of methylglyoxal, (b) the specific base catalyzed formation of glutathione-(R,S)-methylglyoxal thiohemiacetals, (c) the glyoxalase I catalyzed conversion of the diastereotopic thiohemiacetals to (S)-D-lactoylglutathione, and (d) the glyoxalase II catalyzed hydrolysis of (S)-D-lactoylglutathione to form D-lactate and glutathione. The model exhibits the following properties under conditions where substrate concentrations are small in comparison to the Km values for the glyoxalase enzymes: The overall rate of conversion of methylglyoxal to D-lactate is primarily limited by the rate of formation of the diastereotopic thiohemiacetals. The hydration of methylglyoxal is kinetically unimportant, since the apparent rate constant for hydration is (approximately 500-10(3))-fold smaller than that for formation of the thiohemiacetals. The rate of conversion of methylglyoxal to (S)-D-lactoylglutathione is near optimal, on the basis that the apparent rate constant for the glyoxalase I reaction (kcatEt/Km congruent to 4-20 s-1 for pig, rat, and human erythrocytes) is roughly equal to the apparent rate constant for decomposition of the thiohemiacetals to form glutathione and methylglyoxal [k(obsd) = 11 s-1, pH 7]. The capacity of glyoxalase I to use both diastereotopic thiohemiacetals, versus only one of the diastereomers, as substrates represents a 3- to 6-fold advantage in the steady-state rate of conversion of the diastereomers to (S)-D-lactoylglutathione.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concanavalin A increases glyoxalase enzyme activities in polymorphonuclear leukocytes and lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.     

Quantitative assessment of the glyoxalase pathway in Leishmania infantum as a therapeutic target by modelling and computer simulation

The glyoxalase pathway of Leishmania infantum was kinetically characterized as a trypanothione-dependent system. Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates. A K(m) of 0.253 mm and a V of 0.21 micromol.min(-1).mg(-1)for glyoxalase I, and a K(m) of 0.098 mm and a V of 0.18 micromol.min(-1).mg(-1) for glyoxalase II, were obtained. Modelling and computer simulation were used for evaluating the relevance of the glyoxalase pathway as a potential therapeutic target by revealing the importance of critical parameters of this pathway in Leishmania infantum. A sensitivity analysis of the pathway was performed using experimentally validated kinetic models and experimentally determined metabolite concentrations and kinetic parameters. The measurement of metabolites in L. infantum involved the identification and quantification of methylglyoxal and intracellular thiols. Methylglyoxal formation in L. infantum is nonenzymatic. The sensitivity analysis revealed that the most critical parameters for controlling the intracellular concentration of methylglyoxal are its formation rate and the concentration of trypanothione. Glyoxalase I and II activities play only a minor role in maintaining a low intracellular methylglyoxal concentration. The importance of the glyoxalase pathway as a therapeutic target is very small, compared to the much greater effects caused by decreasing trypanothione concentration or increasing methylglyoxal concentration.     

Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.     

Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A review

Methylglyoxal is a toxic metabolite unavoidably produced in mammalian systems as a by-product of glycolysis. Detoxification of this compound occurs principally through the glyoxalase pathway, which consists of glyoxalase I and glyoxalase II, and requires reduced glutathione as a co-enzyme. Recently, it has been demonstrated that variations in glucose, glutamine and fetal bovine serum levels can cause significant changes in the intracellular concentration of methylglyoxal. More importantly, comparative studies involving wild-type Chinese hamster ovary cells and clones overexpressing glyoxalase I indicate that glucose and glutamine, within the range normally found in cell culture media, can cause decreased cell viability mediated solely through increased production of methylglyoxal. In addition, endogenously produced methylglyoxal has been shown to cause apoptosis in cultured HL60 cells. While the exact mechanism of the impact of methylglyoxal on cultured cells is unknown, methylglyoxal is a potent protein and nucleic acid modifying agent at physiological concentrations and under physiological conditions. Protein modification occurs mainly at arginine, lysine and cysteine residues and is believed to be an important signal for the degradation of senescent proteins. Modification of arginine and lysine results in the irreversible formation of advanced glycation endproducts, whereas modification of cysteine results in the formation of a highly reversible hemithioacetal. Methylglyoxal also forms adducts with nucleic acids, principally with guanyl residues. At high extracellular concentrations, it is genotoxic to cells grown in culture. Even at physiological concentrations (100 nM free methylglyoxal), methylglyoxal can modify unprotected plasmid DNA and cause gene mutation and abnormal gene expression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modification of the glyoxalase system during the functional activation of human neutrophils

The glyoxalase system catalyses the metabolism of methylglyoxal to D-lactic acid, via the intermediate S-D-lactoylglutathione. It is present in human neutrophils and undergoes a significant modification during functional activation--induction of chemotaxis, phagocytosis and degranulation. During the activation of neutrophils with serum-opsonised zymosan and the tumour-promoting phorbol diester 12-O-tetradecanoylphorbol 13-acetate, the activity of glyoxalase I increases and the activity of glyoxalase II decreases by 20-40% of their activities in resting cells, in the initial 10 min of the activation period. Determination of the Michaelis constant, Km, and the apparent maximum velocity, Vmax, for these enzymatic reactions indicates that the change in activity is due to a non-competitive activation and inhibition of glyoxalase I and glyoxalase II, respectively. This is consistent with a modification of the glyoxalase enzyme protein during the activation response. This modification occurs under aerobic and anaerobic incubation conditions. The concentration of S-D-lactoylglutathione increases approx. 100% of the resting cell concentration during the initial 10 min of the activation period. The presence of S-D-lactoylglutathione in neutrophils may be related to its ability to stimulate microtubule assembly.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

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.     

Passive cigarette smoke and renal glyoxalase system

Cigarette smoking is associated with a number of fatal diseases, including cancer of different organs. A number of oxoaldehydes are found in cigarette smoke, among which methylglyoxal (MG) is known to cause toxicity to cells upon accumulation. In biological systems, MG is converted to s-d-lactoylglutathione by glyoxalase I with reduced glutathine (GSH) as a cofactor, and s-d-lactoylglutathione is converted to D-lactic acid with simultaneous regeneration of GSH, by glyoxalase II. In the present study, we have investigated the status of the glyoxalase enzymes in kidney tissues from rats exposed to passive cigarette smoke. No significant change has been noted in glyoxalase I activity. Glyoxalase II was decreased during 1 and 2 weeks of exposure, and after that the activity was increased. The initial decrease in the activity of gly II may be due to the excess amount of methylglyoxal generated due to smoke exposure or the adduct formed by MG and GSH which known to inhibit gly II activity. Both enzymes help in the detoxification of cigarette smoke induced chemicals and biochemicals.     

2-Oxoaldehyde metabolism in microorganisms

The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes. The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate. The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase. The regulation of the yeast glyoxalase system was analyzed. The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones. The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied.