Abstract
Methylglyoxal(MG) is a potent cytotoxin that is produced as a byproduct of various metabolic reactions in the cell. The major enzymes for MG detoxification are Glyoxalase I(GLYI), Glyoxalase II(GLYII) and D-lactate dehydrogenase(D-LDH). These three enzymes work together and convert MG into D-pyruvate, which directly goes to TCA cycle. Here, a comparative study of the ability of MG detoxification of these three enzymes has been done in both E. coli and yeast. Ectopic expression of these three genes from Arabidopsis in E. coli in presence of different abiotic stress revealed the contribution of each of these genes in detoxifying MG. Yeast mutants of MG detoxification enzymes were also grown in different stress conditions to record the effect of each gene. These mutants were also used for complementation assays using the respective MG detoxifying genes from Arabidopsis in presence of various stress conditions. The MG content and the corresponding growth of cells was measured in all the bacterial as well as yeast strains. This study reveals differential contribution of MG detoxification enzymes in mitigating MG levels and alleviating stress in both prokaryotes as well as eukaryotes. GLYI and D-LDH were found to be key enzymes in MG detoxification under various abiotic stresses.
Highlights
Methylglyoxal (MG) is a three carbon metabolite which is present in all the organisms from prokaryotes to eukaryotes
BL21 cells transformed with pET28a-AtGLYI, pET28a-AtGLYII, pET28a-AtD-LDH and empty vector pET28a were grown in presence of different abiotic stresses and their growth was monitored spectrophotometrically
In response to all the stresses under study, E. coli cells overexpressing Arabidopsis genes for MG detoxification enzymes grew better than the cells transformed with empty vector and accumulated lower MG levels than the cells with empty vector
Summary
Methylglyoxal (MG) is a three carbon metabolite which is present in all the organisms from prokaryotes to eukaryotes. Glyoxalase pathway is the major system for MG detoxification in all the organisms including bacteria, yeast, humans, plants and animals It comprises of two enzymes, Glyoxalase I (GLYI) which converts MG to S-D-lactoylglutathione (SLG) and Glyoxalase II (GLYII) which converts SLG to D-lactate[29]. A NADH and NADPH dependent methylglyoxal reductase has been identified in E.coli which converts methylglyoxal directly to acetol (Misra et al 1996) The overexpression of these aldo-keto reductases in plants have been found to confer tolerance to various stresses. Another enzyme, MG dehydrogenase converts MG into pyruvate. Results of stress tolerance and complementation assays were validated by measuring endogenous MG level in wild type as well as mutant cells grown in presence and absence of stress to correlate the effect of each gene in reducing MG level and congruent tolerance to abiotic stress
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