Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence

Metallo-β-lactamases (MBLs) are class B β-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of β-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Interplay among Oxidative Stress, Methylglyoxal Pathway and S-Glutathionylation

Reactive oxygen species (ROS) are produced constantly inside the cells as a consequence of nutrient catabolism. The balance between ROS production and elimination allows to maintain cell redox homeostasis and biological functions, avoiding the occurrence of oxidative distress causing irreversible oxidative damages. A fundamental player in this fine balance is reduced glutathione (GSH), required for the scavenging of ROS as well as of the reactive 2-oxoaldehydes methylglyoxal (MGO). MGO is a cytotoxic compound formed constitutively as byproduct of nutrient catabolism, and in particular of glycolysis, detoxified in a GSH-dependent manner by the glyoxalase pathway consisting in glyoxalase I and glyoxalase II reactions. A physiological increase in ROS production (oxidative eustress, OxeS) is promptly signaled by the decrease of cellular GSH/GSSG ratio which can induce the reversible S-glutathionylation of key proteins aimed at restoring the redox balance. An increase in MGO level also occurs under oxidative stress (OxS) conditions probably due to several events among which the decrease in GSH level and/or the bottleneck of glycolysis caused by the reversible S-glutathionylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase. In the present review, it is shown how MGO can play a role as a stress signaling molecule in response to OxeS, contributing to the coordination of cell metabolism with gene expression by the glycation of specific proteins. Moreover, it is highlighted how the products of MGO metabolism, S-D-lactoylglutathione (SLG) and D-lactate, which can be taken up and metabolized by mitochondria, could play important roles in cell response to OxS, contributing to cytosol-mitochondria crosstalk, cytosolic and mitochondrial GSH pools, energy production, and the restoration of the GSH/GSSG ratio. The role for SLG and glyoxalase II in the regulation of protein function through S-glutathionylation under OxS conditions is also discussed. Overall, the data reported here stress the need for further studies aimed at understanding what role the evolutionary-conserved MGO formation and metabolism can play in cell signaling and response to OxS conditions, the aberration of which may importantly contribute to the pathogenesis of diseases associated to elevated OxS.

Genomic identification, Characterization and Expression Analysis of Glyoxalase Gene Family in Brassica Napus L.

Background Glyoxalase I (GLYI) and glyoxalase II (GLYII), two enzymes of the glyoxalase pathway, are responsible for the detoxification of a cytotoxic metabolite methylglyoxal (MG) into the nontoxic S-D-lactoylglutathione, which play crucial roles in stress tolerance in various plant species. Considering the roles of glyoxalases, the GLY gene families have been analyzed in higher plant, such as rice, soybean and Chinese cabbage; however, little is known about them in rapeseed (Brassica napus L.). The B. napus glyoxalase pathway is worth an in-depth investigation about the presence, distribution, localizations, and expression of glyoxalase genes. Result In this study, a total of 35 BnaGLYI and 30 BnaGLYII genes were identified in the B. napus genome, and they were clustered into six and eight subfamilies, respectively. The classifications, chromosomal distributions, gene structures, and conserved motifs were predicted and analyzed. Importantly, these genes were mainly localized in chloroplast and cytoplasm. Moreover, their expression profiles varied among different tissues. For example, BnaGLY genes are expressed in most organs but tend to be highly expressed in a single organ, such as BnaGLYI27 and BnaGLYII19; while some members are only expressed in specific tissues, e.g., BnaGLYI32 only expressed in silique pericarps. Some genes were induced at different germination stages. Notably, a number of BnaGLY genes showed responses to Plasmodiophora brassicae infection. Conclusion This study systematically identifies BnaGLYI and BnaGLYII gene families in B. napus. The different sub-cellular organelles and expression analysis offer insight into their biological roles and function in plant development and stress resistance.

Protection of Polyphenols against Glyco-Oxidative Stress: Involvement of Glyoxalase Pathway

Chronic high glucose (HG) exposure increases methylglyoxal (MGO)-derived advanced glycation end-products (AGEs) and is involved in the onset of pathological conditions, such as diabetes, atherosclerosis and chronic-degenerative diseases. Under physiologic conditions the harmful effects of MGO are contrasted by glyoxalase system that is implicated in the detoxification of Reactive Carbonyl Species (RCS) and maintain the homeostasis of the redox environment of the cell. Polyphenols are the most abundant antioxidants in the diet and present various health benefits. Aims of the study were to investigate the effects of HG-chronic exposure on glyco-oxidation and glyoxalase system in intestinal cells, using CaCo-2 cells. Moreover, we studied the effect of apple polyphenols on glyco-oxidative stress. Our data demonstrated that HG-treatment triggers glyco-oxidation stress with a significant increase in intracellular Reactive Oxygen Species (ROS), lipid peroxidation, AGEs, and increase of Glyoxalase I (GlxI) activity. On the contrary, Glyoxalase II (GlxII) activity was lower in HG-treated cells. We demonstrate that apple polyphenols exert a protective effect against oxidative stress and dicarbonyl stress. The increase of total antioxidant capacity and glutathione (GSH) levels in HG-treated cells in the presence of apple polyphenols was associated with a decrease of GlxI activity.

Protection of Polyphenols Against Glyco-Oxidative Stress: Involvement of Glyoxalase Pathway

Chronic high glucose (HG) exposure increases methylglyoxal (MG)-derived AGEs and is involved in the onset of pathological conditions, such as diabetes, atherosclerosis and chronic‐degenerative diseases. Under physiologic condition the harmful effects of MG are contrasted by glyoxalase system that is involved in the detoxification of Reactive Carbonyl Species (RCS) and maintain the homeostasis of the redox environment of the cell. Polyphenols are the most abundant antioxidants in the diet and present various health benefits. The study aimed at investigating the role of polyphenols extracted from an apple high in polyphenols (Calville White Winter), on glyco-oxidative stress induced by chronic HG-exposure. Intestinal Caco-2 cells were treated in physiological glucose condition (25mM) as a control and in HG condition (50mM) with or without apple extract for one week. Our data demonstrated that HG-treatment triggers glyco-oxidation stress with a significantly increase in ROS, lipid peroxidation, AGEs and Glyoxalase I (GlxI) activity with a significant decrease in total antioxidant intracellular defense. Treatment with polyphenols under HG condition restores to the control levels GlxI activity, decreases Glyoxalase II (GlxII) in relation to the control and induces a drop of glyco-oxidative damage. This paper seeks to highlight the roles of polyphenols in glyco-oxidative stress.

Antimicrobial prodrug activation by the staphylococcal glyoxalase GloB

ABSTRACTWith the rising prevalence of multidrug-resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g. serum half-life, oral absorption, solubility, toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating “prodrugs” that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, S. schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologs are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species-or genus-level prodrug targeting strategies.

Preliminary Characterization of a Ni2+-Activated and Mycothiol-Dependent Glyoxalase I Enzyme from Streptomyces coelicolor

The glyoxalase system consists of two enzymes, glyoxalase I (Glo1) and glyoxalase II (Glo2), and converts a hemithioacetal substrate formed between a cytotoxic alpha-ketoaldehyde, such as methylglyoxal (MG), and an intracellular thiol, such as glutathione, to a non-toxic alpha-hydroxy acid, such as d-lactate, and the regenerated thiol. Two classes of Glo1 have been identified. The first is a Zn2+-activated class and is exemplified by the Homo sapiens Glo1. The second class is a Ni2+-activated enzyme and is exemplified by the Escherichia coli Glo1. Glutathione is the intracellular thiol employed by Glo1 from both these sources. However, many organisms employ other intracellular thiols. These include trypanothione, bacillithiol, and mycothiol. The trypanothione-dependent Glo1 from Leishmania major has been shown to be Ni2+-activated. Genetic studies on Bacillus subtilis and Corynebacterium glutamicum focused on MG resistance have indicated the likely existence of Glo1 enzymes employing bacillithiol or mycothiol respectively, although no protein characterizations have been reported. The current investigation provides a preliminary characterization of an isolated mycothiol-dependent Glo1 from Streptomyces coelicolor. The enzyme has been determined to display a Ni2+-activation profile and indicates that Ni2+-activated Glo1 are indeed widespread in nature regardless of the intracellular thiol employed by an organism.

Exogenous application of gibberellic acid mitigates drought-induced damage in spring wheat

Drought stress is a major problem in wheat production but it could be managed by using various exogenous protectants such as gibberellic acid (GA). Although GA is a plant growth hormone, it shows a potential to protect the plant in stress conditions. To investigate the possible role of GA in mitigating drought stress, we treated wheat (<em>Triticum aestivum</em> ‘BARI Gom-21’) seedlings with a GA spray under semihydroponic conditions. In the experiment, the combined effect of GA and drought stress (induced by 12% polyethylene glycol) was studied after 48 h and 72 h. In the absence of exogenous GA, drought-stressed wheat seedlings showed various physiological and biochemical changes in a time-dependent manner. Malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and free proline (Pro) concentrations were increased, whereas catalase (CAT) and ascorbate peroxidase (APX) activities were reduced under drought stress. Gibberellic acid played a role in restoring the ascorbate (AsA) level, decreased the reduced/oxidized glutathione (GSH/GSSG) ratio and reduced monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) activities. Gibberellic acid significantly affected the glyoxalase system. Under drought stress, the methylglyoxal (MG) concentration was increased but GA application stimulated glyoxalase I (Gly I) and glyoxalase II (Gly II) activities to protect the wheat seedlings against stress. The study concluded that the severity of drought stress in wheat depends on the growth stage and it increases with an increase in the duration of stress, whereas exogenous GA helped the seedlings to survive by upregulating antioxidant defense mechanisms and the glyoxalase system.