The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1

Abstract

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.

Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

“…For example, the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inhibited by GSSG, H 2 O 2 and diamide (Holtgrefe et al, 2008; Zaffagnini et al, 2013) and α-amylase and starch synthase, are regulated by disulfide-bond formation (Seung et al, 2013; Skryhan et al, 2015). Due to the central role of AtTPIs in carbon metabolism, we tested whether redox agents may have an effect on cTPI and pdTPI catalytic activities.…”

StTrxF, a potato plastidic thioredoxin F-type protein gene, is involved in starch accumulation in transgenic Arabidopsis thaliana

“…Starch, as a main source of nutrition in human and animal diet, is a major storage carbohydrate in plants, which plays a fundamentally important role in plant survival and adaptation to varying environmental conditions [1,2]. In plants, starch is an insoluble glucan composed of two glucose polymers: amylose and amylopectin.…”

“…Starch metabolism is regulated at many levels, including allosteric regulation by redox regulation, proteinprotein interactions and protein phosphorylation [2,6]. Redox regulation, which has been widely researched in plant starch biosynthesis, is also related to the coordination of enzymes involved in starch degradation [6,7].…”

“…Trxs are essential players in the regulation of multiple cellular processes [11,12]. The active site of Trxs contains a conserved CXXC motif with two reactive cysteine (Cys) residues, which is located within the characteristic thioredoxin fold [2]. Since the plastidic Trxs F-type proteins act in vitro as reductants for a number of the redox-regulated enzymes involved in starch metabolism, it has been suggested that the redox regulation that occurs in response to light signals is similar to that controlling other photosynthetic enzymes [7,13].…”

Constitutive Expression of <i>VvAATP</i> Increases Starch Content in Transgenic <i>Arabidopsis</i>

“…As a main source of nutrition in the human and animal diet, starch is a major storage of carbohydrates in plants [3], and plays a fundamental role in plant survival and adaptation to various environmental conditions [4]. In plants, starch exists as an insoluble glucan, comprised of two glucose polymers: amylose and amylopectin.…”