Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.     

Glyoxalase II from Zea mays: properties and inhibition study of the enzyme purified by use of a new affinity ligand

The synthesis of N-(p-nitrocarbobenzoxy)glutathione (N-pNCBG) is reported. N-pNCBG and glutathione (GSH) were coupled to Affi-gel 10 by a thioester linkage and resulted in very effective bound ligands for a fast purification of glyoxalase II from corn. The S-(N-pNCBG)-affinity column showed a glyoxalase II binding capacity of up to 2-fold higher than that of the glutathione-affinity column. A single form of glyoxalase II was evidenced by PAGE in both crude extracts and in the affinity purified enzyme. A 45% recovery of glyoxalase II activity (purification, approx. 433-fold) was obtained for both matrices by a single chromatography. The purified glyoxalase is an acidic protein (pI 4.5) of about 26,000 relative molecular mass. Substrate studies for the corn glyoxalase II show, among possible substrates tested, that S-D-lactyl-glutathione is the preferred substrate. An inhibition study was performed with methyl-, propyl-, hexyl-, p-nitrobenzyl-, p-chlorophenacyl-, carbobenzoxy-, and p-nitrocarbobenzoxy-S-glutathione. Methyl-S-glutathione did not inhibit corn glyoxalase II; the others were found to be linear competitive inhibitors. The derivatives containing a thioether bond are weaker inhibitors than those containing a thioester bond or a carbonyl group. p-Nitrobenzyl-S-glutathione is the weakest inhibitor; the carbobenzoxy-S-derivatives are stronger inhibitors than the p-chlorophenacyl S-derivative.     

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.     

Role of rpoS in the regulation of glyoxalase III in Escherichia coli

Methylglyoxal is an endogenous electrophile produced in Escherichia coli by the enzyme methylglyoxal synthase to limit the accumulation of phosphorylated sugars. In enteric bacteria methylglyoxal is detoxified by the glutathione-dependent glyoxalase I/II system, by glyoxalase III, and by aldehyde reductase and alcohol dehydrogenase. Here we demonstrate that glyoxalase III is a stationary-phase enzyme. Its activity reached a maximum at the entry into the stationary phase and remained high for at least 20 h. An rpoS- mutant displayed normal glyoxalase I and II activities but was unable to induce glyoxalase III in stationary phase. It thus appears that glyoxalase III is regulated by rpoS and might be important for survival of non-growing E. coli cultures.     

Yeast glyoxalase I is a monomeric enzyme with two active sites

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.     

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.     

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.     

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.     

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.     

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.     

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.     

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.     

Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II

Earlier we have shown the role of glyoxalase overexpression in conferring salinity tolerance in transgenic tobacco. We now demonstrate the feasibility of same in a crop like rice through overproduction of glyoxalase II. The rice glyoxalase II was cloned in pCAMBIA1304 and transformed into rice (Oryza sativa cv PB1) via Agrobacterium. The transgenic plants showed higher constitutive activity of glyoxalase II that increased further upon salt stress, reflecting the upregulation of endogenous glyoxalase II. The transgenic rice showed higher tolerance to toxic concentrations of methylglyoxal (MG) and NaCl. Compared with non-transgenics, transgenic plants at the T1 generation exhibited sustained growth and more favorable ion balance under salt stress conditions. Sneh L. Singla-Pareek and Sudesh Kumar Yadav have contributed equally to this work.     

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)...     

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.     

Relative distribution of glutathione transferase, glyoxalase I and glyoxalase II in helminths

Glutathione transferase, glyoxalase I and glyoxalase II activities were not evenly distributed among the major helminth groups. Intestinal cestodes and digeneans had higher glutathione transferase activity than parasitic nematodes. High glyoxalase II activity was found in cestodes and digeneans but no glyoxalase I was detectable. Glyoxalase I and II were both detected in nematodes. These results are discussed in relation to the enzymes' suggested role in protection against secondary lipid peroxidation products.     

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.     

Glyoxalase II of African trypanosomes is trypanothione-dependent

The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.