The glyoxalase pathway is ubiquitously found in all the organisms ranging from prokaryotes to eukaryotes. It acts as a major pathway for detoxification of methylglyoxal (MG), which deleteriously affects the biological system in stress conditions. The first important enzyme of this system is Glyoxalase I (GLYI). It is a metalloenzyme which requires divalent metal ions for its activity. This divalent metal ion can be either Zn2+ as found in most of eukaryotes or Ni2+ as seen in prokaryotes. In the present study, we have found three active GLYI enzymes (AtGLYI2, AtGLYI3 and AtGLYI6) belonging to different metal activation classes coexisting in Arabidopsis thaliana. These enzymes have been found to efficiently complement the GLYI yeast mutants. These three enzymes have been characterized in terms of their activity, metal dependency, kinetic parameters and their role in conferring tolerance to multiple abiotic stresses in E. coli and yeast. AtGLYI2 was found to be Zn2+ dependent whereas AtGLYI3 and AtGLYI6 were Ni2+ dependent. Enzyme activity of Zn2+ dependent enzyme, AtGLYI2, was observed to be exceptionally high (~250–670 fold) as compared to Ni2+ dependent enzymes, AtGLYI3 and AtGLYI6. The activity of these GLYI enzymes correlated well to their role in stress tolerance. Heterologous expression of these enzymes in E. coli led to better tolerance against various stress conditions. This is the first report of a higher eukaryotic species having multiple active GLYI enzymes belonging to different metal activation classes.
Abiotic stress and climate change is the major concern for plant growth and crop yield. Abiotic stresses lead to enhanced accumulation of reactive oxygen species (ROS) consequently resulting in cellular damage and major losses in crop yield. One of the major scavengers of ROS is ascorbate (AA) which acts as first line of defense against external oxidants. An enzyme named ascorbate oxidase (AAO) is known to oxidize AA and deleteriously affect the plant system in response to stress. Genome-wide analysis of AAO gene family has led to the identification of five, three, seven, four, and six AAO genes in Oryza sativa, Arabidopsis, Glycine max, Zea mays, and Sorghum bicolor genomes, respectively. Expression profiling of these genes was carried out in response to various abiotic stresses and during various stages of vegetative and reproductive development using publicly available microarray database. Expression analysis in Oryza sativa revealed tissue specific expression of AAO genes wherein few members were exclusively expressed in either root or shoot. These genes were found to be regulated by both developmental cues as well as diverse stress conditions. The qRT-PCR analysis in response to salinity and drought stress in rice shoots revealed OsAAO2 to be the most stress responsive gene. On the other hand, OsAAO3 and OsAAO4 genes showed enhanced expression in roots under salinity/drought stresses. This study provides lead about important stress responsive AAO genes in various crop plants, which could be used to engineer climate resilient crop plants.
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.
Steroids are a group of organic compounds that include sex hormones, adrenal cortical hormones, sterols, and phytosterols. In mammals, steroid biosynthesis starts from cholesterol via multiple steps to the final steroid and occurs in the gonads, adrenal glands, and placenta. This highly regulated pathway involves several cytochrome P450, as well as different dehydrogenases and reductases. Steroids in mammals have also been associated with drug production. Steroid pharmaceuticals such as testosterone and progesterone represent the second largest category of marketed medical products. There heterologous production through microbial transformation of phytosterols has gained interest in the last couple of decades. Phytosterols being the plants sterols serve as inexpensive substrates for the production of steroid derivatives. Various genes and biochemical pathways involved in phytosterol degradation have been identified in many Rhodococcus and Mycobacterium species. Apart from an early investigation in mammals, presence of steroids such as androsteroids and progesterone has also been demonstrated in plants. Their main role is linked with growth, development, and reproduction. Even though plants share some chemical features with mammals, the biosynthesis is different, with the first C22 hydroxylation as an example. This is performed by CYP11A1 in mammals and CYP90B1 in plants. Moreover, the entire plant steroid biosynthesis is not fully elucidated. Knowing this pathway could provide new processes for the industrial biotechnological production of steroid hormones in plants.
Glyoxalase pathway is the major pathway of methylglyoxal detoxification and is ubiquitously present in all organisms ranging from prokaryotes to eukaryotes. Glyoxalase I (GLYI) and Glyoxalase II (GLYII), the two core enzymes of this pathway work together to neutralize methylglyoxal (MG), a dicarbonyl molecule with detrimental cytotoxicity at higher concentrations. The first step towards the detoxification of MG is catalyzed by GLYI, a metalloenzyme that requires divalent metal ions (either Zn 2+ as seen in eukaryotes or Ni 2+ as in prokaryotes). However, both Zn 2+ and Ni 2+ dependent GLYIs have been shown to co-exist in a higher eukaryote i.e. Arabidopsis thaliana. In the present study, we determine the role of both Zn 2+ dependent (AtGLYI2) and Ni 2+ dependent (AtGLYI3, AtGLYI6) GLYIs from Arabidopsis in salinity stress tolerance. AtGLYI2 overexpressing Arabidopsis plants showed better growth rate while maintaining lower levels of MG under high saline conditions. They were taller with more number of silique formation with respect to their Ni 2+ dependent counterparts. Further, lack in germination of Arabidopsis AtGLYI2 mutants in presence of exogenous MG indicates the direct involvement of Zn 2+ dependent GLYI in MG detoxification, suggesting Zn 2+ dependent GLYI as the main enzyme responsible for MG detoxification and salinity stress tolerance.
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