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.     

Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant

Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.     

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.     

Bacterial glyoxalase enzymes

The glyoxalase system is composed of two metalloenzymes, Glyoxalase I and Glyoxalase II. This system is important in the detoxification of methylglyoxal, among other roles. Detailed studies have determined that a number of bacterial Glyoxalase I enzymes are maximally activated by Ni(2+) and Co(2+) ions, but are inactive in the presence of Zn(2+). This is in contrast to the Glyoxalase I enzyme from humans, which is catalytically active with Zn(2+) as well as a number of other metal ions. The structure-activity relationships between these two classes of Glyoxalase I are serving as important clues to how the molecular structures of these proteins control metal activation profiles as well as to clarify the mechanistic chemistry of these catalysts. In addition, the possibility of targeting inhibitors against the bacterial versus human enzyme has the potential to lead to new approaches to combat bacterial infections.     

Purification and characterization of glyoxalase I from Hansenula mrakii

Glyoxalase I was purified from Hansenula mrakii IFO 0895 which was resistant to 25 mM methylglyoxal. The molecular weight of the purified enzyme was calculated to be 38,000 by both gel-filtration of Sephadex G-150 and SDS-PAGE. The enzyme was almost specific to methylglyoxal (K_m = 0.91 mM). The activity of the enzyme was not inhibited by metal ion chelators such as EDTA, which is a potent inhibitor for glyoxalase Is from other sources.     

Upregulation of Glyoxalase I fails to Normalize Methylglyoxal Levels: A Possible Mechanism for Biochemical Changes in Diabetic Mouse Lenses

Glyoxalase I is the first enzyme in a two-enzyme glyoxalase system that metabolizes physiological methylglyoxal (MGO). MGO reacts with proteins to form irreversible adducts that may lead to crosslinking and aggregation of lens proteins in diabetes. This study examined the effect of hyperglycemia on glyoxalase I activity and its mRNA content in mouse lens epithelial cells (mLE cells) and in diabetic mouse lenses and investigated the relationship between GSH and MGO in organ cultured lenses. mLE cells cultured with 25 mM D-glucose (high glucose) showed an upregulation of glyoxalase I activity and a higher content of glyoxalase I mRNA when compared with either cells cultured with 5 mM glucose (control) or with 20 mM L-glucose + 5 mM D-glucose. MGO concentration was significantly elevated in cells cultured with high D-glucose, but not in L-glucose. GSH levels were lower in cells incubated with high glucose compared to control cells. Glyoxalase I activity and mRNA levels were elevated in diabetic lenses compared to non-diabetic control mouse lenses. MGO levels in diabetic lenses were higher than in control lenses. Incubation of lenses with buthionine sulfoximine (BSO) resulted in a dramatic decline in GSH but the MGO levels were similar to lenses incubated without BSO. Our data suggest that in mouse lenses MGO accumulation may occur independent of GSH concentration and in diabetes there is an upregulation of glyoxalase I, but this upregulation is inadequate to normalize MGO levels, which could lead to MGO retention and chemical modification of proteins.     

DNA-uptake in the naturally competent cyanobacterium, Synechocystis sp. PCC 6803

Abstract Eight species of halophilic Archaea were tested for the presence of the enzymes of the methylglyoxal bypass. Methylglyoxal synthase was found in extracts of all species tested, with the exception of Halobacterium salinarium and Halobacterium cutirubrum . The enzyme of Haloferax volcanii was most active at pH 7 in the absence of salt, and in the presence of 3 M NaCl or KCl activity was half of that without salt, and was inhibited by phosphate. Glyoxalase I was detected in all species tested. Optimal activity of H. volcanii glyoxalase I was found at pH 7 and 3 M KCl; in the absence of salt, activity was strongly reduced. Glutathione could be replaced by γ-glutamylcysteine as the acceptor of the D-lactoyl group. The work shows that the methylglyoxal bypass may be operative in representatives of the archaeal kingdom.     

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.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

Lethal synthesis of methylglyoxal by Escherichia coli during unregulated glycerol metabolism

In Escherichia coli K-12, the conversion of glycerol to triose phosphate is regulated by two types of control mechanism: the rate of synthesis of glycerol kinase and the feedback inhibition of its activity by fructose-1,6-diphosphate. A strain which has lost both control mechanisms by successive mutations, resulting in the constitutive synthesis of a glycerol kinase no longer sensitive to feedback inhibition, can produce a bactericidal factor from glycerol. This toxic factor has been identified by chemical and enzymological tests as methylglyoxal. Methylglyoxal can be derived from dihydroxyacetone phosphate through the action of an enzyme which is present at high constitutive levels in the extracts of the mutant as well as that of the wild-type strain. Nine spontaneous mutants resistant to 1 mm exogenous methylglyoxal have been isolated. In all cases the resistance is associated with increased levels of a glutathione-dependent enzymatic activity for the removal of methylglyoxal. Methylglyoxal-resistant mutants derived from the glycerol-sensitive parental strain also became immune to glycerol.     

Sexual response in Saccharomyces cerevisiae: alteration of enzyme activity in the glyoxalase system by mating factor

Glyoxalase I activity in alpha-type budding yeast of the Saccharomyces cerevisiae strain was increased by exposure of alpha-type cells to supernatant of a culture of a-type yeast cells, although glyoxalase II activity was decreased by the same treatment. The alteration of enzyme activity in the glyoxalase system occurred during the 30-60 min period after exposure of alpha-type cells to a-type culture supernatant. No change of glyoxalase I and II activities was found in the case of the alpha-type strain, S. cerevisiae VQ3 (alpha ste3-1), which is deficient in a-factor receptors.     

Salt- and glyphosate-induced increase in glyoxalase I activity in cell lines of groundnut (Arachis hypogaea)

Glyoxalase I (EC 4.4.1.5) activity has long been associated with rapid cell proliferation, but experimental evidence is forthcoming, linking its role to stress tolerance as well. Proliferative callus cultures of groundnut ( Arachis hypogaea L. cv. JL24) showed a 3.3-fold increase in glyoxalase I activity during the logarithmic growth phase, correlating well with the data on FW gain and mitotic index. Inhibition of cell division decreased glyoxalase I activity and vice versa, thus further corroborating its role as a cell division marker enzyme. Cell lines of A. hypogaea selected in the presence of high salt (NaCl) and herbicide (glyphosate) concentrations, yielded 4.2- to 4.5-fold and 3.9- to 4.6-fold elevated glyoxalase I activity, respectively, in a dose dependent manner reflective of the level of stress tolerance. The stress-induced increase in enzyme activity was also accompanied by an increase in the glutathione content. Exogenous supplementation of glutathione could partially alleviate the growth inhibition of callus cultures induced by methylglyoxal and d -isoascorbic acid, but failed to recover the loss in glyoxalase I activity due to d -isoascorbic acid. The adaptive significance of elevated glyoxalase I activity in maintaining glutathione homeostasis has been discussed in view of our understanding on the role of glutathione in the integration of cellular processes with plant growth and development under stress conditions.     

Growth inhibition of Proteus mirabilis by cyclic adenosine 3''-5''-monophosphate.

Growth of Proteus mirabilis on a synthetic agar medium containing either glycerol, galactose, or trehalose as the sole source is inhibited by 5 mM cyclic adenosine 3',5'-monophosphate (cAMP). Inhibition on an agar medium is evident as loss of viability, but in broth cAMP only slightly inhibits growth rate. Inhibition is associated with the accumulation of methylglyoxal in the medium. A nonswarming mutant of P. mirabilis is not inhibited by cAMP on either of the three carbon sources, but it is sensitive to exogenous methylglyoxal.     

Unique regulation of glyoxalase I activity during osmotic stress response in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>: neither the mRNA nor the protein level of glyoxalase I increase under conditions that enhance its activity

Glyoxalase I is a ubiquitous enzyme that catalyzes the conversion of methylglyoxal, a toxic 2-oxoaldehyde derived from glycolysis, to S-D-lactoylglutathione. The activity of glyoxalase I in the fission yeast Schizosaccharomyces pombe was increased by osmotic stress induced by sorbitol. However, neither the mRNA levels of its structural gene nor its protein levels increased under the same conditions. Cycloheximide blocked the induction of glyoxalase I activity in cells exposed to osmotic stress. In addition, glyoxalase I activity was increased in stress-activated protein kinase-deficient mutants (wis1 and spc1). We present evidence for the post-translational regulation of glyoxalase I by osmotic stress in the fission yeast.     

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.     

A Glutathione-independent Glyoxalase of the DJ-1 Superfamily Plays an Important Role in Managing Metabolically Generated Methylglyoxal in Candida albicans

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys¹³⁶-His¹³⁷-Glu¹⁶⁸. Purified Glx3p has an in vitro methylglyoxalase activity (K_m = 5.5 mm and k_cat = 7.8 s⁻¹) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.     

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.     

Purification and characterization of glyoxalase I from Pseudomonas putida

Glyoxalase I was purified to apparent homogeneity from Pseudomonas putida. The enzyme was a monomer with a molecular weight of 20,000. The enzyme was most active at pH 8.0. The Km values for methylglyoxal and 4,5-dioxovale-rate are 3.5 mM and 1.2 mM, respectively. Contrary to the case of eukaryotic enzymes, chelating agents showed little inhibitory effects on the enzyme activity. Among the metal ions tested, Zn++ specifically and completely inhibited the activity of the enzyme at a millimolar level. The properties of bacterial glyoxalase I were quite different from mammalian and yeast enzymes.     

Novel approach for structural identification of protein family: glyoxalase I

Glyoxalase is one of two enzymes of the glyoxalase detoxification system against methylglyoxal and other aldehydes, the metabolites derived from glycolysis. The glyoxalase system is found almost in all living organisms: bacteria, protozoa, plants, and animals, including humans, and is related to the class of ‘life essential proteins’. The enzyme belongs to the expanded Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily. At present the GenBank contains about 700 of amino acid sequences of this enzyme type, and the Protein Data Bank includes dozens of spatial structures. We have offered a novel approach for structural identification of glyoxalase I protein family, which is based on the selecting of basic representative proteins with known structures. On this basis, six new subfamilies of these enzymes have been derived. Most populated subfamilies A1 and A2 were based on representative human Homo sapiens and bacterial Escherichia coli enzymes. We have found that the principle feature, which defines the subfamilies’ structural differences, is conditioned by arrangement of N- and C-domains inside the protein monomer. Finely, we have deduced the structural classification for the glyoxalase I and assigned about 460 protein sequences distributed among six new subfamilies. Structural similarities and specific differences of all the subfamilies have been presented. This approach can be used for structural identification of thousands of the so-called hypothetical proteins with the known PDB structures allowing to identify many of already existing atomic coordinate entrees.