The role of cysteine residues in redox regulation and protein stability of <i>Arabidopsis thaliana</i> starch synthase 1

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed

Redox Regulation of Starch Metabolism

Metabolism of starch is a major biological integrator of plant growth supporting nocturnal energy dynamics by transitory starch degradation as well as periods of dormancy, re-growth, and reproduction by utilization of storage starch. Especially, the extraordinarily well-tuned and coordinated rate of transient starch biosynthesis and degradation suggests the presence of very sophisticated regulatory mechanisms. Together with the circadian clock, land plants (being autotrophic and sessile organisms) need to monitor, sense, and recognize the photosynthetic rate, soil mineral availability as well as various abiotic and biotic stress factors. Currently it is widely accepted that post-translational modifications are the main way by which the diel periodic activity of enzymes of transient starch metabolism are regulated. Among these mechanisms, thiol-based redox regulation is suggested to be of fundamental importance and in chloroplasts, thioredoxins (Trx) are tightly linked up to photosynthesis and mediate light/dark regulation of metabolism. Also, light independent NADP-thioredoxin reductase C (NTRC) plays a major role in reactive oxygen species scavenging. Moreover, Trx and NTRC systems are interconnected at several levels and strongly influence each other. Most enzymes involved in starch metabolism are demonstrated to be redox-sensitive in vitro. However, to what extent their redox sensitivity is physiologically relevant in synchronizing starch metabolism with photosynthesis, heterotrophic energy demands, and oxidative protection is still unclear. For example, many hydrolases are activated under reducing (light) conditions and the strict separation between light and dark metabolic pathways is now challenged by data suggesting degradation of starch during the light period