Biosynthesis Regulation Of Gene Expression And Enzyme Activities

Hell Really Exists

Hell Really Exists

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The pathway of glutathione biosynthesis is well established and is similar in plants, animals and micro-organisms. In two ATP-dependent steps, catalysed by y-glutamylcysteine synthetase (y-ECS) and glutathione synthetase (GS), the constituent amino acids are linked to form the complete tripeptide. The N-terminal peptide bond linking glutamic acid to cysteine in gSh is unusual in that glutamic acid is linked via the than the group. The two-step reaction sequence occurs in both chloroplastic and non-chloroplastic compartments and is found in photosynthetic and non-photosynthetic tissues (Law and Halliwell 1986, Klapheck et al. 1987, Hell and Bergmann 1988, 1990, Ruegsegger and Brunold 1993, Noctor et al. 1998a, Noctor and Foyer 1998). The existence of a mitochondrial isoform of y-ECS in Brassica juncea has been postulated (Schäfer et al. 1998).

In maize, which has strong demarcation of metabolism within photosynthetic cells, including antioxidants (Doulis et al. 1997), cysteine is synthesized in the bundle sheath whereas GS activity is located predominantly in the mesophyll cells (Burgener et al. 1998). It appears, therefore, that glu-tathione is synthesized in the cells where GR is present and that the bundle sheath relies on the mesophyll for the synthesis of glutathione and the reduction of GSSG. At the subcellular level, the distribution of enzyme activity between chloroplast and extra-chloroplastic compartments is approximately equal. Chloroplasts from Pisum sativum contained 72 % of leaf y-ECS activity and 48 % of leaf GS activity (Klapheck et al. 1987, Hell and Bergmann 1990). In Spinacia oleracea, values of 61 % (y-ECS) and 48 % (GS) were found (Hell and Bergmann 1990). The very low activities of these enzymes in plants and the complexities of the procedures for enzyme extraction and assay have precluded extensive purification and kinetic characterization. Consequently, much of our current knowledge of their structure, regulation and function has been gleaned from molecular techniques and plant transformation, and to date this remains limited.

The gene encoding y-ECS here denoted as gshl, was originally cloned from Arabidopsis thaliana by complementation of an E. coli mutant deficient in this enzyme (May and Leaver 1994). Heterologous expression of the Arabidopsis y-ECS in a yeast mutant only recovered 10 % of the GSH measured in the wild-type yeast (May and Leaver 1994). This discrepancy provoked much speculation concerning the identity of the cloned gene, but further complementation studies have now confirmed that this gene does indeed encode a protein with true y-ECS activity (May et al. 1998a).

Functional complementation of an E. coli mutant deficient in GS activity was also used to clone the Arabidopsis thaliana gene for this enzyme, which we denote here as gsh2 (Rawlins et al. 1995, Ullman et al. 1996) The ability of several plant species to make analogues of glutathione depends on the specificity of the synthetases involved. Specific legume GSs use either glycine to form GSH or ß-alanine to form homoglutathione (MacNicol 1987). Recent evidence from Medicago truncatula suggests that separate genes encode GS and homoglutathione synthetase (hGS) and that the divergence in specificity has arisen by gene duplication after the evolutionary divergence of the Fabaceaea (Frendo et al. 1999). The two genes are very homologous and are found on the same fragment of genomic DNA (Mathieu 1999).

Relatively little is known about the co-ordinate regulation of expression of gshl and gsh2 but it is clear that GSH and GSSGper se have no control over the expression of these genes (Xiang and Oliver 1998). Similarly, H202 did not affect expression. The abundance of gsh1 and gsh2 transcripts was increased by cadmium in Brassica juncea (Schäfer et al. 1998). Xiang and Oliver (1998) found that both cadmium and copper increased transcript abundance in this species (Figure 3). Interestingly, JA also increased gsh1 and gsh2 transcripts and a common signal transduction pathway may be involved (Xiang and Oliver 1998). Most importantly, oxidative stress was required for the translation of the transcripts in these conditions, suggesting that post-transcriptional regulation was involved in the control of GSH synthesis and that H202 (or low GSH/GSSG ratios) de-represses translation of the existing mRNA (Xiang and Oliver 1998). Studies in animals, particularly on cancer cells challenged with chemotherapeutic agents, have shown that transcription of the gene is regulated by protein factors and by con served antioxidant response elements upstream of the coding sequence (Figure 3).

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