%0 Generic
%A Gromes, Roland
%D 2007
%F heidok:7948
%K RedoxregulationRedox regulation
%R 10.11588/heidok.00007948
%T Post-translational regulation and evolution of plant gamma-glutamate cysteine ligase
%U https://archiv.ub.uni-heidelberg.de/volltextserver/7948/
%X Glutamate cysteine ligase (GCL) is catalyzing the rate-limiting step in glutathione (GSH) synthesis. A complex regulation of this enzyme is required to integrate various signals as GSH is fulfilling a plethora of functions in housekeeping metabolism, stress defence, and in the regulation of development. In this thesis the post-translational redox regulation of plant GCL and closely related proteobacterial enzymes was studied. The crystal structure of Brassica juncea GCL (BjGCL) revealed the presence of two intramolecular disulfide bridges. Biochemical analyses of the wild-type enzyme and of mutants lacking cysteines required for the formation of either disulfide bridge showed that both bridges are involved in the in vitro redox regulation of BjGCL. One disulfide bridge (CC1) is apparently controlling access to the active site and knock-out results in a slower overall catalysis rate without changes in Km-values. The second disulfide bridge (CC2) controls the formation of a GCL homo-dimer and reduction of this disulfide bridge leads to monomerization and almost complete deactivation of the enzyme. Sequence analysis showed that only CC2 is conserved in all higher plants while the occurrence of CC1 is restricted to the Rosids clade. Characterization of the redox regulation of GCL from the (non-Rosid) Nicotiana tabacum confirmed the presence of only the dimerization-dependent mechanism of redox regulation. Furthermore, it could be shown that feedback-inhibition of plant GCL by GSH is mechanistically independent from redox regulation. A model is presented on how these different mechanisms interact to control GSH synthesis in vivo. Comparative sequence analysis of plant GCL and with related enzymes from proteobacteria revealed that the amino acid residues forming the dimer interface in BjGCL are conserved in higher plants only, while the catalytic residues are highly conserved among all sequences. The characterization of recombinantly produced GCL from Agrobacterium tumefaciens and Xanthomonas campestris confirmed that these enzymes show kinetics and susceptibility to inhibitors similar to the plant enzyme but completely lack redox regulation and are active as monomers. In a second project, the influence of soluble thiols on the GSH metabolism of different types of cultured plant cells was studied, revealing a specific induction of GCL expression by cysteine. This observation may hint at a role of GSH synthesis in the control of the cellular concentrations of this amino acid, preventing an accumulation which might lead to oxidative stress.