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Methylglyoxal is the most important intracellular glycation agent, formed nonenzymatically from triose phosphates during glycolysis in eukaryotic cells. Methylglyoxal-derived advanced glycation end-products are involved in neurodegenerative disorders (Alzheimer's, Parkinson's and familial amyloidotic polyneurophathy) and in the clinical complications of diabetes. Research models for investigating protein glycation and its relationship to methylglyoxal metabolism are required to understand this process, its implications in cell biochemistry and their role in human diseases. We investigated methylglyoxal metabolism and protein glycation in Saccharomyces cerevisiae. Using a specific antibody against argpyrimidine, a marker of protein glycation by methylglyoxal, we found that yeast cells growing on d-glucose (100 m m) present several glycated proteins at the stationary phase of growth. Intracellular methylglyoxal concentration, determined by a specific HPLC based assay, is directly related to argpyrimidine formation. Moreover, exposing nongrowing yeast cells to a higher d-glucose concentration (250 m m) increases methylglyoxal formation rate and argpyrimidine modified proteins appear within 1 h. A kinetic model of methylglyoxal metabolism in yeast, comprising its nonenzymatic formation and enzymatic catabolism by the glutathione dependent glyoxalase pathway and aldose reductase, was used to probe the role of each system parameter on methylglyoxal steady-state concentration. Sensitivity analysis of methylglyoxal metabolism and studies with gene deletion mutant yeast strains showed that the glyoxalase pathway and aldose reductase are equally important for preventing protein glycation in Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

Glyoxalase I is a ubiquitous enzyme that catalyzes the conversion of methylglyoxal, a toxic 2-oxoaldehyde derived from glycolysis, toS-D-lactoylglutathione. The activity of glyoxalase I in the fission yeastSchizosaccharomyces pombewas 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 (wis1andspc1). We present evidence for the post-translational regulation of glyoxalase I by osmotic stress in the fission yeast. [ABSTRACT FROM AUTHOR]

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 Δglo1 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 kcat/Km 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. [ABSTRACT FROM AUTHOR]

The present study regards possible changes in the activity of glyoxalase system enzymes (glyoxalase I, GI, and glyoxalase II, GII) in tissues (brain, liver and white muscle) of the mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of Cu in the marine water and on control untreated animals. The experiments also included measurements of copper concentration in the tissues, as well as of lactate dehydrogenase (LDH) activity, to evaluate possible Cu accumulation and changes in glycolytic activity respectively. Cu accumulation only occurs in the liver. GI, GII and LDH activities kept unchanged in the brain after copper exposure. GI activity in liver and muscle of copper-exposed animals decreases probably for a slackening in the glycolytic rate, as suggested by the lowering of LDH activity. GII activity remains unchanged or increases (liver extract, 0.5 ppm of Cu), maybe to safeguard enough cellular levels of GSH. [Copyright &y& Elsevier]

Three-dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term '3D domain swapping' was first used to describe the dimeric structure of diphtheria toxin, the database of domain-swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain-swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain-swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain-swapped proteins, but also a framework for understanding future findings of 3D domain swapping. [ABSTRACT FROM AUTHOR]

A role of glyoxalase I (Gly-I), a detoxifying enzyme, in cell viability of prostate cancer was investigated. Cell extracts obtained from 66 prostate tissue specimens and prostatic cancer PC-3 cells were assayed for Gly-I activity using the spectrophotometric method. Gly-I activity was consistently more than eightfold higher in prostate cancer (CAP) specimens (n=37) than in non-cancerous (NCP) specimens (n=29). To understand the importance of such a high Gly-I activity in CAP specimens, the effects of methylglyoxal (MG) on PC-3 cells were examined in vitro. MG, a putative toxic glycolytic metabolite, was capable of inducing severe (>99%) cell death in 24 h, along with a significant reduction in activities of Gly-I as well as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), a key glycolytic enzyme. However, such severe cell death was effectively (~85%) prevented with N-acetylcysteine (NAC), a precursor of reduced glutathione (GSH) that is an essential cofactor for Gly-I, accompanied by the intact Gly-I and G3PDH activities. Therefore, Gly-I may play a critical detoxifying role in glycolysis to maintain cellular activity and viability of prostatic cancer cells. [ABSTRACT FROM AUTHOR]

The present study was performed to investigate the effect of oltipraz on passive smoke-induced alteration in renal glyoxalase system of rats. Adult Sprague-Dawley rats were exposed daily to passive cigarette smoke in a whole-body exposure chamber 6 h per day for 2, 4 and 12 weeks. The animals being sacrificed after 2 and 12 weeks were maintained on control diet, powdered 4% Teklad rat chow (Harlan Teklad, Madison, WI, USA). The 4 weeks group was divided into three subgroups, one receiving control diet, other two receiving control diet supplemented with two doses of oltipraz (either 167 or 500 ppm), starting 1 week prior to initiation of smoke exposure until the end of the experiment. The activity of glyoxalase I was higher in animals exposed for 4 and 12 weeks of passive smoke than those exposed for 2 weeks. There was no significant difference between 4 and 12 weeks. Glyoxalase II activity was lower in animals exposed to passive smoke for 4 weeks than those exposed for 2 weeks. However, the activity approached the basal level after 12 weeks of exposure. Furthermore, oltipraz treatment maintained the activity of both glyoxalase closer to the basal levels. [ABSTRACT FROM AUTHOR]

The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program hyperfit. Glyoxalase I exhibited saturation kinetics on 0.05–2.5 mm hemithioacetal concentration range, with kinetic parameters Km 0.53 ± 0.07 mm and V (3.18 ± 0.16) × 10-2 mm·min-1. Glyoxalase II also showed saturation kinetics in the Sd-lactoylglutathione concentration range of 0.15–3 mm and Km 0.32 ± 0.13 mm and V (1.03 ± 0.10) × 10-3 mm·min-1 were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program gepasi. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis–Menten model. The kinetic parameters obtained, Km 0.62 ± 0.18 mm and V (2.86 ± 0.01) × 10-2 mm·min-1, were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis–Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed. [ABSTRACT FROM AUTHOR]

The present work aimed to study the activities of glyoxalase system enzymes, glyoxalase I (G I) and glyoxalase II (G II), as well as the expression of their genes in human breast carcinoma. Samples of tumoral tissue and normal counterparts were drawn from several patients during surgery. They served either for preparing extracts to be used in enzyme activity evaluations or for RNA extraction and subsequent northern blot analysis. A far higher activity level of G I and G II occurs in the tumor compared with pair-matched normal tissue, as shown by both spectrophotometrical assay and electrophoretic pattern. Such increased activities of G I and G II likely result from an enhanced enzyme synthesis as a consequence of increased expression of the respective genes in the tumoral tissue, as evidenced by northern blot. The present findings confirm a key-role of glyoxalase system to detoxify cytotoxic methylglyoxal and modulate S-D-lactoylglutathione levels in tumor cells. Moreover, they suggest a possible employment of GI inhibitors as anti-cancer drugs. [ABSTRACT FROM AUTHOR]

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme. [ABSTRACT FROM AUTHOR]

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglu-tathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO. [ABSTRACT FROM AUTHOR]

Com o objetivo de se estudar a susceptibilidade genética à paracoccidioidomicose- infecçâo, procurou-se determinar uma possível associação entre a glioxalase 1 e a reação intradérmicaàparacoccidioidina. O fenótipo GLO 1 ocorreu em freqüência significativamente mais alta entre os indivíduos com reação positiva.With the putpose to study the genetic susceptibility to paracoccidioidomycosis-infection we searched for a possible association between glyoxalase I and the intradermic paracoccidioidin reaction. The phenotype GLO 1 was significantly more frequent among positive reactors.

Methylglyoxal (MG) is a cytotoxic compound often produced as a side product of metabolic processes such as glycolysis, lipid peroxidation, and photosynthesis. MG is mainly scavenged by the glyoxalase system, a two-step pathway, in which the coordinate activity of GLYI and GLYII transforms it into D-lactate, releasing GSH. In Arabidopsis thaliana, a member of the GLYI family named GLYI4 has been recently characterized. In glyI4 mutant plants, a general stress phenotype characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness was observed. In order to shed some light on the impact of gly4 loss-of-function on plant metabolism, we applied a high resolution mass spectrometry-based metabolomic approach to Arabidopsis Col-8 wild type and glyI4 mutant plants. A compound library containing a total of 70 metabolites, differentially synthesized in glyI4 compared to Col-8, was obtained. Pathway analysis of the identified compounds showed that the upregulated pathways are mainly involved in redox reactions and cellular energy maintenance, and those downregulated in plant defense and growth. These results improved our understanding of the impacts of glyI4 loss-of-function on the general reprogramming of the plant's metabolic landscape as a strategy for surviving under adverse physiological conditions. [ABSTRACT FROM AUTHOR]

The dermis is the connective layer between the epidermis and subcutis and harbours nerve endings, glands, blood vessels, and hair follicles. The most abundant cell type is the fibroblast. Dermal fibroblasts have a versatile portfolio of functions within the dermis that correspond with different types of cells by either direct contact or by autocrine and paracrine signalling. Diabetic skin is characterized by itching, numbness, ulcers, eczema, and other pathophysiological changes. These pathogenic phenotypes have been associated with the effects of the reactive glucose metabolite methylglyoxal (MGO) on dermal cells. In this study, dermal fibroblasts were isolated from diabetic and non-diabetic human donors. Cultured dermal fibroblasts from diabetic donors exhibited reduced insulin-induced glucose uptake and reduced expression of the insulin receptor. This diabetic phenotype persists under cell culture conditions. Secretion of IL-6 was increased in fibroblasts from diabetic donors. Increased secretion of IL-6 and MIF was also observed upon the treatment of dermal fibroblasts with MGO, suggesting that MGO is sufficient for triggering these immunomodulatory responses. Remarkably, MIF treatment resulted in decreased activity of MGO-detoxifying glyoxalase-1. Given that reduced glyoxalase activity results in increased MGO levels, these findings suggested a positive-feedback loop for MGO generation, in which MIF, evoked by MGO, in turn blocks MGO-degrading glyoxalase activity. Finally, secretion of procollagen Type I C-Peptide (PICP), a marker of collagen production, was reduced in fibroblast from diabetic donors. Remarkably, treatment of fibroblasts with either MGO or MIF was sufficient for inducing reduced PICP levels. The observations of this study unravel a signalling network in human dermal fibroblasts with the metabolite MGO being sufficient for inflammation and delayed wound healing, hallmarks of T2D. [ABSTRACT FROM AUTHOR]

The zinc metalloenzyme glyoxalase I catalyses the glutathione­dependent inactivation of toxic methylglyoxal. The structure of the dimeric human enzyme in complex with S-benzyl-glutathione has been determined by multiple isomorphous replacement (MIR) and refined at 2.2 Å resolution. Each monomer consists of two domains. Despite only low sequence homology between them, these domains are structurally equivalent and appear to have arisen by a gene duplication. On the other hand, there is no structural homology to the ‘glutathione binding domain’ found in other glutathione­linked proteins. 3D domain swapping of the N- and C-terminal domains has resulted in the active site being situated in the dimer interface, with the inhibitor and essential zinc ion interacting with side chains from both subunits. Two structurally equivalent residues from each domain contribute to a square pyramidal coordination of the zinc ion, rarely seen in zinc enzymes. Comparison of glyoxalase I with other known structures shows the enzyme to belong to a new structural family which includes the Fe2+-dependent dihydroxybiphenyl dioxygenase and the bleomycin resistance protein. This structural family appears to allow members to form with or without domain swapping. [ABSTRACT FROM AUTHOR]

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modem family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions. [ABSTRACT FROM AUTHOR]

The possible effect of several physiologically important aldehydes has been tested on partially purified glyoxalase I of Ehrlich ascites carcinoma (EAC) cells. The results indicate that D, and L-lactaldehyde are strong non-competitive inhibitors of glyoxalase I and the effect with the D-isomer is more pronounced, whereas both D,L-glyceraldehyde and acetaldehyde are moderately inhibitory and the nature of inhibition is strictly competitive. Moreover, D,L-glyceraldehyde strongly inhibits the utilization of methylglyoxal by intact EAC cells. A search for the presence of several aldehyde metabolizing enzymes in EAC cells indicates that non-specific aldehyde reductase, methylglyoxal reductase, aldehyde dehydrogenase and alcohol dehydrogenase are apparently absent in this rapidly growing, highly de-differentiated malignant cell. [ABSTRACT FROM AUTHOR]

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[Display omitted] • The carboxyl group in the TLSC702 molecule is critical for binding to GLO I. • Lipophilicity of TLSC702 derivatives is correlated with antiproliferative effects. • Addition of hydrophobic groups at phenyl position increases apoptosis inducibility. Glyoxalase I (GLO I) is a known therapeutic target in cancer. Even though TLSC702, a GLO I inhibitor that we discovered, induces apoptosis in tumor cells, exceptionally higher doses are required compared with those needed to inhibit GLO I activity in vitro. In this work, structure–activity optimization studies were conducted on four sections of the TLSC702 molecule to determine the partial structural features necessary for the inhibition of GLO I. Herein, we found that the carboxy group in TLSC702 was critical for binding with the divalent zinc at the active site of GLO I. In contrast, the side chain substituents in the meta- and para- positions of the benzene ring had little influence on the in vitro inhibition of GLO I. The CLogP values of the TLSC702 derivatives showed a positive correlation with the antiproliferative effects on NCI-H522 cells. Thus, two derivatives of TLSC702, which displayed either high or low lipophilicity due to the types of substituents at the phenyl position, were selected. Even though both derivatives showed comparable inhibitory effects as that of their parent compound, the derivative with the high CLogP value was distinctly more antiproliferative than TLSC702. In contrast, the derivative with the low CLogP value did not decrease cell viability in NCI-H522 and HL-60 cells. These findings suggested that structural improvements, such as the addition of hydrophobic moieties to the phenyl group, enhanced the ability of TLSC702 to induce apoptosis by increasing cell membrane permeability. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is a typical malignancy and the most common in the female and it is the primary reason behind cancer-related deaths of women around the world. The pathological role of the non-enzymatic change of proteins by reducing sugars become frequently shows in different kinds of cancer. Cancer cells generally rely upon aerobic glycolysis as the main source of energy. Impaired glucose metabolism is somewhat responsible for the aggregation of advanced glycation end products (AGEs). Methylglyoxal (MG), a glycolysis byproduct either contributes to the accumulation of AGEs. Enzymatic defense upon AGEs products exists in all mammalian cells., Aims: The present work intends to look into Glyoxalase1 (GLO1) and fructosamine-3-kinase (FN3K) activity in human breast carcinoma., Methods: Thirty-three consecutive patients were entered into the study. Samples of breast tumoral tissue and normal matches were drawn from patients after surgery. FN3K and GLO1 enzymatic activity were analyzed using a radiometric and spectrophotometric assay., Results: The average level of FN3K enzyme was fundamentally lower in cancerous tissues parallel with adjacent noncancerous tissues. We also observed a consistent increase of GLO1 activity in the tumor parallel with pair-matched normal tissue., Conclusion: The current findings build up a key-role of enzymatic defense to detoxify cytotoxic AGEs and methylglyoxal levels in tumor cells. These discoveries may give another system to the treatment of breast cancer., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© the Author(s).)

ESD (esterase D), GLO1 (glyoxalase I), and GPT (glutamate pyruvate transaminase) are human erythrocytic isoenzymes and have previously been applied in forensic medicine caseworks. The molecular bases of the polymorphic gene expression products have been demonstrated to be because of SNPs in respective coding regions. However, it has not been revealed whether the SNPs conferring the polymorphisms to the aforementioned erythrocytic isoenzymes could be simultaneously detected by using a simple PCR method. In this study, we used mismatched primers to simultaneously amplify three common isoenzyme loci so that all amplified products contained the same Hph I cleavage sites. The products were then digested with Hph I and electrophoretically separated and stained so that alleles were identified. The accumulated values for the probability of discrimination power and excluding the probability of paternity to the aforementioned systems attained 90.41% and 41.72%, respectively, in the Chinese Han population. This assay could be extremely valuable for future forensic medicine practices. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MG), a reactive dicarbonyl compound mainly produced by metabolic pathways, such as glycolysis, binds to proteins or nucleic acids and forms advanced glycation end products. MG is efficiently metabolized by the glyoxalase system where MG is converted by glyoxalase I (GLO I) to S-D-lactoylglutathione. Although the glyoxalase system has been shown to play a pathological role in various diseases, including diabetic complications, its detailed pathophysiological function remains to be elucidated. We are interested in renal hypoxic diseases, but very little information is available regarding the association between the glyoxalase system and renal hypoxic diseases. Therefore, we investigated the biological role of GLO I in renal hypoxic diseases by using the rat ischemia/reperfusion (I/R) injury model. I/R induced the reduction of renal GLO I activity associated with morphological changes and renal dysfunction. Interestingly, the rats that overexpress human GLO I (GLO I Tg rats) showed amelioration of these manifestations in renal I/R (e.g., improvement of the tubulointerstitial injury and renal function). Accumulation of renal MG adducts, carboxyethyllysine, induced by I/R also decreased in GLO I Tg rats compared to wild-type rats. These results demonstrate that GLO I has renoprotective effects in I/R injury via reduction of protein modification by MG. [ABSTRACT FROM AUTHOR]

• Four different QM-clusters were used to study the catalytic reaction of Glyoxalase I. • A QM-cluster with a small Glu-99 can produce the mechanism of the S substrate. • The same cluster predicts relative stabilities of the substrate's binding modes. • The same cluster cannot produce the mechanism of the R substrate. • It is better to use QM/MM methods to study reactions of stereospecific enzymes. Quantum mechanical (QM) cluster approach is a useful and convenient tool to study mechanisms of enzymatic reactions. Our investigation is to propose a proper QM-cluster that reproduces the special specificity of glyoxalase I (GlxI). GlxI converts S and R enantiomers of hemithioacetal to only S -D enantiomer of its product. It shows this special specificity with two glutamate residues that are symmetrically coordinated to a Zn ion. However, molecular dynamics simulations showed that one of the glutamates, Glu-172 is more flexible than the other one, Glu-99. Results indicate that a QM-cluster with a more flexible model of Glu-172 can reproduce proposed mechanisms for the S -substrate. However, the same cluster can reach to the R -L enantiomer of the product from the R substrate. We propose the necessity of using much more expensive, hybrid QM/MM methods or a very big QM-cluster to model and study reaction mechanisms of stereospecific enzymes like GlxI. [ABSTRACT FROM AUTHOR]

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of GlyoxalaseI (Gly I). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I. This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba (Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus, ID: KT720495.1; Brassica juncea ID: Y13239.1, Arachis hypogaea; ID: DQ989209.2; and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I, when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I (Jojo-Gly I) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I, confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants. [ABSTRACT FROM AUTHOR]

Yellow mosaic virus (YMV) infection occurred through whitefly (Bemisia tabaci) transmitted begomovirus in black gram during kharif season but the crop did not show any signs of infection during the spring season. To compare and comprehend the intricate link between the glutathione molecule and methylglyoxal detoxification pathway, three black gram cultivars-cv.KUG253 (highly susceptible to YMV), MASH114 (resistant) and cross (cv. KUG253 × MASH114) were sown during kharif and spring season. YMV brutally infected the leaves of cv.KUG253 in the kharif season at 20 days after sowing (DAS) and caused severe chlorosis by 40 DAS which made the leaves papery and feeble. On the contrary, YMV infection was completely absent in the other two cultivars of the black gram and during the spring season, all three cultivars exhibited normal growth without any sign of this disease. The main cytotoxic product formed under YMV infection was methylglyoxal, as significantly higher specific activity was observed for methylglyoxal reductase (MGR) and glyoxalase enzymes in the kharif season rather than spring season in all the three cultivars. Glyoxalase III has been recently discovered for the detoxification of MG and is independent of the glutathione molecule, but no report has been published till now which elaborately explains its role during the pathogenic attack on the plants. • Positive correlation of g-glutamylcysteine synthetase with glyoxalase I and glyoxalase II during YMV infection. • Upregulation of glutathione peroxidase and glutathione-s-transferase during YMV infection in black gram. • Lower activity of methylglyoxal reducrase resulted in accumulation of methylglyoxal during YMV infection. • Glyoxalase III activity was independent of the presence of reduced glutathione. [ABSTRACT FROM AUTHOR]

Members of the Brassicaceae family have the ability to regulate pollination events occurring on the stigma surface. In Brassica species, self-pollination leads to an allele-specific interaction between the pollen small cysteine-rich peptide ligand (SCR/SP11) and the stigmatic S-receptor kinase (SRK) that activates the E3 ubiquitin ligase ARC1 (Armadillo repeat-containing 1), resulting in proteasomal degradation of various compatibility factors including glyoxalase I (GLO1) which is necessary for successful pollination. In Brassica napus, the suppression of GLO1 was sufficient to reduce compatibility, and overexpression of GLO1 in self-incompatible Brassica napus stigmas resulted in partial breakdown of the self-incompatibility response. Here, we verified if BnGLO1 could function as a compatibility factor in the artificial self-incompatibility system of Arabidopsis thaliana expressing AlSCR b , AlSRK b and AlARC1 proteins from A. lyrata. Overexpression of BnGLO1 is sufficient to breakdown self-incompatibility response in A. thaliana stigmas. Therefore, GLO1 has an indisputable role as a compatibility factor in the stigma in regulating pollen attachment and pollen tube growth. Lastly, this study demonstrates the usefulness of an artificial self-incompatibility system in A. thaliana for interspecific self-incompatibility studies.

The accumulation of a metabolic by product - methylglyoxal above a minimal range can be highly toxic in all organisms. Stress induced elevation in methylglyoxal inactivates proteins and nucleic acids. Glutathione dependent glyoxalase enzymes like glyoxalase I and glyoxalase II together with glutathione independent glyoxalase III play inevitable role in methylglyoxal detoxification. Glyoxalase genes are generally conserved but with obvious exceptions. Mangroves being potent harsh land inhabitants, their internal organelles are constantly been exposed to elevated levels of methylglyoxal. First and foremost it is important to detect the presence of glyoxalases in mangroves. De novo transcriptome analysis of mangrove species Rhizophora mucronata Lam., identified eleven putative glyoxalase proteins (RmGLYI-1 to 5, RmGLYII-1 to 5 and RmGLYIII). Molecular characterization proposed PLN02300 or PLN02367 as the key domains of RmGLYI proteins. They possess molecular weight ranging from 26.45 to 32.53 kDa and may localize in cytosol or chloroplast. RmGLYII proteins of molecular weight 28.64-36 kDa, carrying PLN02398 or PLN02469 domains are expected to be localized in diverse cellular compartments. Cytosolic RmGLYIII with DJ-1/PfpI domain carries a molecular weight 26.4 kDa. Detailed structural analysis revealed monomeric nature of RmGLYI-1 and RmGLYII-1 whereas RmGLYIII is found to be homodimer. Molecular phylogenetic analysis and multiple sequence alignment specified conserved metal ion/substrate binding residues of RmGLY proteins. Estimation of relative expression of glyoxalases under salt stress indicated the prominence of RmGLYI and RmGLYII over RmGLYIII. The aforementioned prominence is supported by salt induced expression difference of glutathione metabolic enzymes and glutathione regulated transporter protein., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of Glyoxalase I ( Gly I ). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I . This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba ( Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus , ID: KT720495.1; Brassica juncea ID: Y13239.1, Arachis hypogaea ; ID: DQ989209.2; and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I , when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I ( Jojo-Gly I ) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I , confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants.

• Anabaena sp. PCC7120 genome possesses only Alr2321 as a Ni2+dependent active glyoxalase I enzyme like other prokaryotes. • Alr2321 is a key enzyme of Anabaena sp. PCC7120 for toxic methylglyoxal (MG) detoxification. • alr2321 transcript was induced under a range of abiotic stresses. • Alr2321 provides tolerance to cells against various abiotic stresses through detoxification of MG and reactive oxygen species. Abiotic stresses enhance the cellular level of reactive oxygen species (ROS) which consequently leads to toxic methylglyoxal (MG) production. Glyoxalases (GlyI & GlyII) catalyze the conversion of toxic MG into non-toxic lactic acid but their properties and functions have been overlooked in cyanobacteria. This is the first attempt to conduct a genome-wide analysis of GlyI protein (PF00903) from Anabaena sp. PCC7120. Out of total nine GlyI domain possessing proteins, only three (Alr2321, Alr4469, All1022) harbour conserve His/Glu/His/Glu metal binding site at their homologous position and are deficient in conserved region specific for Zn2+ dependent members. Their biochemical, structural and functional characterization revealed that only Alr2321 is a homodimeric Ni2+ dependent active GlyI with catalytic efficiency 11.7 × 106 M−1 s−1. It has also been found that Alr2321 is activated by various divalent metal ions and has maximum GlyI activity with Ni2+ followed by Co2+ > Mn2+ > Cu2+ and no activity with Zn2+. Moreover, the expression of alr2321 was found to be maximally up-regulated under heat (19 fold) followed by cadmium, desiccation, arsenic, salinity and UV-B stresses. BL21/pGEX-5X2- alr2321 showed improved growth under various abiotic stresses as compared to BL21/pGEX-5X2 by increased scavenging of intracellular MG and ROS levels. Taken together, these results suggest noteworthy links between intracellular MG and ROS, its detoxification by Alr2321, a member of GlyI family of Anabaena sp. PCC7120, in relation to abiotic stress. [ABSTRACT FROM AUTHOR]

Advanced glycation end products (AGEs) play an important role in the onset of diabetic retinopathy. Therefore, in the current study, we investigate whether and how Tanshinone IIa (Tan IIa) from Salvia miltiorrhiza protects bovine retinal endothelial cells (BRECs) against methylglyoxal (MGO) mediated cell dysfunction. The results showed that MGO reduced cell viability in dose dependent manner. The treatment of Tan IIa (50 μM) significantly improved cell viability induced by MGO in BRECs. MGO increased cellular reactive oxygen species formation and cellular nitric oxide (NO) level; enhanced nox1 and iNOS mRNA levels; inhibited prdx1 mRNA level. The treatment of Tan IIa effectually ameliorated cellular oxidative stress. Exposure of MGO resulted in mitochondrial fission and decrease of opa1 and mfn1. No significant difference in mRNA levels of mfn2 and drp1 was detected between MGO and medium. Tan IIa reduced mitochondrial fragmentation, enhanced the mRNA levels of mfn1 and opa1 in MGO cultured BRECs. The short time exposure of cellular antioxidatants, dimethylthiourea (10 mM) and tiron (10 mM) had no effect on mitochondrial fission although they ameliorated cellular reactive oxygen species level. Moreover, overexpression of glyoxalase 1 (GLO1) increased key proteins of mitochondrial fusion, including opa1 and mfn1 in BRECs cultured with MGO. However, inhibition of GLO1 by siRNA abolished the effect of Tan IIa on induction of mitochondrial fusion in MGO cultured BRECs. In conclusion, MGO caused the injury of retinal endothelial cells through induction of mitochondrial dysfunction and mitochondrial fission, the treatment of Tan IIa ameliorated mitochondrial dysfunction and fission induced by AGEs through enhancing GLO1. [ABSTRACT FROM AUTHOR]

Aging is a complex natural phenomenon that is manifested by degenerative changes in the structure and function of cells and tissues. D-Galactose-induced aging mice are an artificial accelerated aging model that causes memory and learning impairment, oxidative stress, and neuroinflammation. In this study, we examined the underlying mechanism of an aging mouse model induced by D-galactose. Our behavioral Morris water maze results revealed that D-galactose administration for 2 months significantly induced memory and learning impairment in C57BL/6J mice. High performance liquid chromatography (HPLC) results showed elevated levels of the metabolite methylglyoxal (MG) in D-galactose-induced aging mice. Whether and how D-galactose induces senescence by elevated levels of reactive metabolite MG remain unclear. In our study, MG mainly accumulated through the following two aspects: to increase its source, namely, the triose phosphate produced by the glycolysis pathway, and to reduce its detoxification system, namely, the glyoxalase system. Therefore, elevated MG levels may be one of the causes of brain senescence in D-galactose-induced mice. However, the molecular mechanism of the increased level of the reaction metabolite methylglyoxal requires further exploration., (Copyright © 2019 Li, Zheng, Chen, Liu and Zhang.)

Detoxification of methylglyoxal, a toxic by-product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d-lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel-shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. DATABASE: Structural data are available in The Protein Data Bank database under the accession numbers 6BNN, 6BNX, and 6BNZ., (© 2019 Federation of European Biochemical Societies.)

Key Message: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.