Thiol-based Control of Tetrapyrrole Metabolism: Posttranslational Control of NADPH-Dependent Thioredoxin Reductase C on Enzymes of Tetrapyrrole Biosynthesis

The NADPH-dependent thioredoxin reductase C (NTRC), thioredoxin and related proteins are involved in thiol/disulfide exchange reactions affecting many key enzymes in central metabolic pathways of photosynthetic organisms. This posttranslational control applies also to tetrapyrrole biosynthesis to fine-tune enzyme activities in response to environmental and endogenous cues and to prevent accumulation of photoreactive tetrapyrrole metabolites. In previous studies several proteins of the tetrapyrrole biosynthetic pathway have been identified to interact with these thiol-reductants. In my group, studies on posttranslational control of tetrapyrrole biosynthesis have been already performed and confirm the regulatory impact of thiol-disulfide exchanges between thioredoxin and NTRC and two proteins, the CHLI subunit of the Mg chelatase (MgCh) complex and the MgProto methyltransferase (CHLM), respectively (Luo et al. 2012, Richter et al. 2013). Reduced content of glutamyl-tRNA reductase (GluTR), CHLM and protochlorophyllide oxidoreductase (POR) has been found in a pale-green Arabidopsis ntrc mutant (Richter et al., 2003) indicating that NTRC is essential for stability and/or activity of enzymes in tetrapyrrole biosynthesis and likely a central redox regulator of tetrapyrrole biosynthesis. The main objective of the proposed project is to identify and validate the thiol-based redox switches of the three enzymes of the tetrapyrrole biosynthetic pathway, CHLM, GluTR and POR, which are catalysed by NTRC, and to elucidate the physiological effects of the posttranslational modifications of these NTRC target enzymes as well as, in consequence, the balanced supply of the end-products, chlorophyll and heme, in the balanced quantities. After confirmation of NTRC interactions with tetrapyrrole biosynthesis enzymes by alternative biochemical methods, it is intended (i) to examine the redox-active thiol switches that control the activity and stability of these enzymes and (ii) to analyse in planta the modulated enzyme activity of Cys>Ser substitution mutants of these enzymes in comparison to wild type always in an Arabidopsis knock-out mutant background for the respective analysed enzyme. It is expected that elucidation of details of the thiol-disulfide exchange reactions on enzymes of tetrapyrrole biosynthesis will improve understanding on redox-controlled tetrapyrrole biosynthesis, which includes changes of enzyme activities, protein structure and stability as well as interaction of proteins to others components of the pathway and their localization within the chloroplast.