Identification of thermostable glyoxalase I in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1⁺) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1⁺ enhanced susceptibility to methylglyoxal, while expression of glo1⁺ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1⁺ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the k_cat/K_m value for methylglyoxal was 4.3×10⁷ M^–1 min^–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h.     

Transgenic potato overproducing L-ascorbic acid resisted an increase in methylglyoxal under salinity stress via maintaining higher reduced glutathione level and glyoxalase enzyme activity

Salt-tolerance was studied in transgenic potato. It was conferred by overexpression of ascorbate pathway enzyme (d-galacturonic acid reductase, GalUR). As genetic engineering of the GalUR gene in potato enhances its ascorbic acid content (l-AsA), and subsequently plants suffered minimal oxidative stress-induced damage, we now report on the comprehensive aptness of this engineering approach for enhanced salt tolerance in transgenic potato (Solanum tuberosum L. cv. Taedong Valley). Potatoes overexpressing GalUR grew and tuberized in continuous presence of 200 mM of NaCl. The transgenic plants maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio together with enhanced activity of glutathione dependent antioxidative and glyoxalase enzymes under salinity stress. The transgenics resisted an increase in methylglyoxal that increased radically in untransformed control plants under salinity stress. This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal (a potent cytotoxic compound) under salt stress. These results suggested the engineering of ascorbate pathway enzymes as a major step towards developing salinity tolerant crop plants.     

Isolation and sequencing of a gene coding for glyoxalase I activity from Salmonella typhimurium and comparison with other glyoxalase I sequences

The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn²⁺ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.     

Mussel glyoxalase I as a possible marker for ecotoxicological studies: Purification and preliminary characterization

Alpha-ketoaldehydes may be formed in cells during oxidative processes and glyoxalase I is the main enzyme involved in the detoxification pathway for these highly toxic compounds. Increased glyoxalase I activity has been observed in mussels exposed to high environmental levels of pollutants and a role for this enzyme as a protection mechanism against peroxidation damage has been hypothesized. In this paper, glyoxalase I from mussel tissue has been purified and a preliminary investigation of its molecular properties carried out. A two step purification procedure for glyoxalase I from digestive gland of Mytilus galloprovincialis is described. The pure enzyme is a 48 kDa protein with an heterodimeric quaternary structure composed of 24 and 25 kDa subunits. The isoelectric point of the native enzyme is at pH 5.0 and there is a divalent cation (probably Zn⁺⁺) requirement for activity. The series of alkyl-S-glutathiones, from methyl- to decyl-, are competitive inhibitors of glyoxalase I. Ki values exponentially decrease from 1.15 mM to 2.65 μM with increasing chain length. Mussel glyoxalase I exhibits molecular properties similar to those of the mammalian enzyme. The possible role of glyoxalase I in the detoxification of α-ketoaldehydes formed during oxidative stress is discussed.     

Oxidative stress and glyoxalase I activity mediate dicarbonyl toxicity in MCF-7 mamma carcinoma cells and a tamoxifen resistant derivative

BackgroundAcquired tamoxifen resistance is a significant problem in estrogen receptor positive breast cancer. In a cellular model, tamoxifen resistance was associated with increased sensitivity towards toxic dicarbonyls and reduced free sulfhydryl group content. We here analyzed the role of oxidative stress and glyoxalase I activity on dicarbonyl resistance and the significance of glyoxalase I expression for survival.MethodsReactive oxygen species were determined by 2,7-dihydrochlorofluorescein diacetate. Inhibitors for NADPH-oxidase (diphenyleneiodonium), p38 MAPK (SB203580) and ERK1/2 (UO126) were applied to investigate interactions of these signaling molecules. N-acetyl cysteine was used to evaluate the effect of oxidative stress on cell viability, which was assessed by the resazurin assay. Gene expression was analyzed by real time qRT-PCR. Glyoxalase activity was inhibited by the specific inhibitor CS-0683 and siRNA. The relevance of glyoxalase 1 mRNA abundance on survival of breast cancer patients was evaluated by the KM-plotter web interface.Resultsα-Oxo-aldehydes caused an immediate increase in reactive oxygen species where the tamoxifen resistant cell line (TamR) responded at lower concentrations than the MCF-7 parental cell line. Inhibitor studies placed ROS production by NADPH-oxidase downstream of p38 MAPK. The antioxidant N-acetyl cysteine (NAC) increased survival, whereas glyoxalase (GLO1) inhibition increased dicarbonyl toxicity. GLO1 mRNA abundance was correlated with unfavorable prognosis of breast cancer patients.ConclusionsDicarbonyl toxicity was mediated by oxidative stress and GLO1 activity determines aldehyde toxicity in tamoxifen resistant cells.General SignificanceGlyoxalases might be predictive biomarkers for tamoxifen resistance and a putative target for the treatment of tamoxifen resistant breast cancer patients.     

Crystal structure of Staphylococcus aureus Zn-glyoxalase I: new subfamily of glyoxalase I family

The crystal structures of protein SA0856 from Staphylococcus aureus in its apo-form and in complex with a Zn²⁺-ion have been presented. The 152 amino acid protein consists of two similar domains with α + β topology. In both crystalline state and in solution, the protein forms a dimer with monomers related by a twofold pseudo-symmetry rotation axis. A sequence homology search identified the protein as a member of the structural family Glyoxalase I. We have shown that the enzyme possesses glyoxalase I activity in the presence of Zn²⁺, Mg²⁺, Ni²⁺, and Co²⁺, in this order of preference. Sequence and structure comparisons revealed that human glyoxalase I should be assigned to a subfamily A, while S. aureus glyoxalase I represents a new subfamily B, which includes also proteins from other bacteria. Both subfamilies have a similar protein chain fold but rather diverse sequences. The active sites of human and staphylococcus glyoxalases I are also different: the former contains one Zn-ion per chain; the latter incorporates two of these ions. In the active site of SA0856, the first Zn-ion is well coordinated by His58, Glu60 from basic molecule and Glu40*, His44* from adjacent symmetry-related molecule. The second Zn3-ion is coordinated only by residue His143 from protein molecule and one acetate ion. We suggest that only single Zn1-ion plays the role of catalytic center. The newly found differences between the two subfamilies could guide the design of new drugs against S. aureus, an important pathogenic micro-organism.     

Paclobutrazol mitigates salt stress in <Emphasis Type="Italic">indica</Emphasis> rice seedlings by enhancing glutathione metabolism and glyoxalase system

Stress-induced methylglyoxal (MG) functions as a toxic molecule, inhibiting plant physiological processes such as photosynthesis and antioxidant defense systems. In the present study, an attempt was made to investigate the MG detoxification through glutathione metabolism in indica rice [Oryza sativa L. ssp. indica cv. Pathumthani 1] under salt stress by exogenous foliar application of paclobutrazol (PBZ). Fourteen-day-old rice seedlings were pretreated with 15 mg L^?1 PBZ foliar spray. After 7 days, rice seedlings were subsequently exposed to 0 (control) or 150 mM NaCl (salt stress) for 12 days. Prolonged salt stress enhanced the production of MG molecules and the oxidation of proteins, leading to decreased activity of glyoxalase enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II). Consequently, the decreased glyoxalase activities were also associated with a decline in reduced glutathione (GSH) content and glutathione reductase (GR) activity. PBZ pretreatment of rice seedlings under salt stress significantly lowered MG production and protein oxidation, and increased the activities of both Gly I and Gly II. PBZ also increased GSH content and GR activity along with the up-regulation of glyoxalase enzymes, under salt stress. In summary, salinity induced a high level of MG and the associated oxidative damage, while PBZ application reduced the MG toxicity by up-regulating glyoxalase and glutathione defense system in rice seedlings.     

Erythrocyte glyoxalase I polymorphism in the owl monkey,Aotus

Three erythrocyte glyoxalase I phenotypes were observed in a sample of 235 karyotypically defined New World owl monkeys, Aotus. The selective distribution of glyoxalase I allele (GLO¹, GLO²) is related to the karyotype of each animal. Owl monkeys with a karyotype VI had an equal distribution of GLO¹ and GLO² genes in the population. Aotus with karyotype II, III, IV, or V had, exclusively, the GLO² allele (expressed as the fast electrophoretic phenotype), in contrast with monkeys with karyotype I or VII, which had only the GLO¹ allele (expressed as the slow electrophoretic phenotype).     

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.     

Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

Proline and betaine provide protection to antioxidant and methylglyoxal detoxification systems during cold stress in <Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze

The aim of this study was to monitor the influence of proline and betaine exposure on antioxidant and methylglyoxal (MG) detoxification system during cold stress in Camellia sinensis (L.) O. Kuntze. Cold stress enhanced MG and lipid peroxidation levels in tea bud (youngest topmost leaf). This increase was resisted upon the exposure of tea bud to proline and betaine. Exposure of tea bud with proline and betaine also help in maintaining thiol/disulfide ratio during cold stress. Proline exposure enhanced glutathione-S-transferase and glutathione reductase (GR) activity, while betaine exposure increased only GR activity during cold stress. Furthermore, effect of proline/betaine was studied on glyoxalase pathway enzymes that are involved in MG detoxification and comprise of two enzymes glyoxalase I and glyoxalase II. Both proline and betaine showed protective effect on glyoxalase I and activating effect on glyoxalase II during cold stress in tea bud. This investigation, therefore, suggest that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.     

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.     

A simplified method for the purification of human red blood cell glyoxalase. I. Characteristics, immunoblotting, and inhibitor studies

Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. TheK _M value for methylglyoxal-glutathione hemithioacetal was 192±8 µM and thek _cat value was 10.9±0.2 × 10⁴ min^–1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had aK _i value of 0.16±0.04 µM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with aK _i value of 3.12±0.88 µM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with aK _i value of 18±8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.     

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.     

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.     

Functional residues on the enzyme active site of glyoxalase I from bovine brain

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.     

The sterochemical configuration of the lactoyl group of S-lactoylglutathionine formed by the action of glyoxalase I from porcine erythrocytes and yeast

S-Lactoylglutatione formed by the reaction between methylglyoxal and glutathione, catalyzed by glyoxalase I, has been isolated by means of gel filtration. The product was analyzed for content of thiolester, thiol, and d- and l-lactate before and after hydrolysis of the thiolester linkage. From the results it is concluded that glyoxalase I from both porcine erythrocytes and yeast stereospecifically transfers hydrogen to form S-d-lactoylglutathione from methylglyoxal and glutathione.     

Identification of glyoxalase 1 polymorphisms associated with enzyme activity

The glyoxalase system and its main enzyme, glyoxalase 1 (GLO1), protect cells from advanced glycation end products (AGEs), such as methylglyoxal (MG) and other reactive dicarbonyls, the formation of which is increased in diabetes patients as a result of excessive glycolysis. MG is partly responsible for harmful protein alterations in living cells, notably in neurons, leading to their dysfunction, and recent studies have shown a negative correlation between GLO1 expression and tissue damage. Neuronal dysfunction is a common diabetes complication due to elevated blood sugar levels, leading to high levels of AGEs. The aim of our study was to determine whether single nucleotide polymorphisms (SNPs) in the GLO1 gene influence activity of the enzyme. In total, 125 healthy controls, 101 type 1 diabetes, and 100 type 2 diabetes patients were genotyped for three common SNPs, rs2736654 (A111E), rs1130534 (G124G), and rs1049346 (5′-UTR), in GLO1. GLO1 activity was determined in whole blood lysates for all participants of the study.