Adenosine-5′-phosphosulfate (APS) kinase (APSK) catalyzes the phosphorylation of APS to 3′-phospho-APS (PAPS). In Arabidopsis thaliana, APSK is essential for reproductive viability and competes with APS reductase to partition sulfate between the primary and secondary branches of the sulfur assimilatory pathway; however, the biochemical regulation of APSK is poorly understood. The 1.8-Å resolution crystal structure of APSR from A. thaliana (AtAPSK) in complex with β,γ-imidoadenosine-5′-triphosphate, Mg 2+ , and APS provides a view of the Michaelis complex for this enzyme and reveals the presence of an intersubunit disulfide bond between Cys86 and Cys119. Functional analysis of AtAPSK demonstrates that reduction of Cys86-Cys119 resulted in a 17-fold higher k cat / K m and a 15-fold increase in K i for substrate inhibition by APS compared with the oxidized enzyme. The C86A/C119A mutant was kinetically similar to the reduced WT enzyme. Gel-and activity-based titrations indicate that the midpoint potential of the disulfide in AtAPSK is comparable to that observed in APS reductase. Both cysteines are invariant among the APSK from plants, but not other organisms, which suggests redox-control as a unique regulatory feature of the plant APSK. Based on structural, functional, and sequence analyses, we propose that the redox-sensitive APSK evolved after bifurcation of the sulfur assimilatory pathway in the green plant lineage and that changes in redox environment resulting from oxidative stresses may affect partitioning of APS into the primary and secondary thiol metabolic routes by having opposing effects on APSK and APS reductase in plants.enzyme kinetics | sulfur metabolism | X-ray crystallography | metabolic evolution S ulfur is an essential element for all living organisms and is required for the biosynthesis of a diverse array of metabolites and macromolecules (1-4). Plants and prokaryotes are the primary assimilatory organisms that convert inorganic sulfate (SO 4 2− ), the predominant form of environmental sulfur, into physiologically useful forms of sulfur (5, 6). The metabolic organization of sulfur assimilation varies among plants and microbes ( Fig. 1) (1-8). In yeast, fungi, and enterobacteria, including Escherichia coli, sulfate is incorporated into adenosine-5′-phosphate (APS), then converted to 3′-phospho-APS (PAPS) as a biologically "activated" compound that is reduced to sulfide (6, 7). In other sulfate-assimilating bacteria, such as Pseudomonas aeruginosa, APS can be used for reduction of sulfide (8). Plants possess bifurcated thiol metabolism pathways, which may reflect the metabolic needs of sessile organisms adapted to a range of environmental stresses and nutrient fluctuations (Fig. 1) (5). These pathways branch after formation of APS. The primary sulfur metabolic route in plants uses APS as the activated highenergy compound for sulfur reduction and the production of cysteine, which is crucial for the synthesis of proteins, methionine, iron-sulfur clusters, vitamin cofactors, and compounds that prote...
Background: CARMIL inhibits the actin capping action of heterodimeric capping protein. Results:Results argue against a steric blocking model and provide evidence for an allosteric mechanism. Conclusion: The conformations of the actin-and CARMIL-binding sites on capping protein are linked. Significance: Control of capping actin barbed ends is critical for cell motility.
Sulfur is essential for plant growth and development, and the molecular systems for maintaining sulfur and thiol metabolism are tightly controlled. From a biochemical perspective, the regulation of plant thiol metabolism highlights nature's ability to engineer pathways that respond to multiple inputs and cellular demands under a range of conditions. In this review, we focus on the regulatory mechanisms that form the molecular basis of biochemical sulfur sensing in plants by translating the intracellular concentration of sulfur-containing compounds into control of key metabolic steps. These mechanisms range from the simple (substrate availability, thermodynamic properties of reactions, feedback inhibition, and organelle localization) to the elaborate (formation of multienzyme complexes and thiol-based redox switches). Ultimately, the dynamic interplay of these regulatory systems is critical for sensing and maintaining sulfur assimilation and thiol metabolism in plants.
Sulfur is an essential plant nutrient and is metabolized into the sulfur-containing amino acids (cysteine and methionine) and into molecules that protect plants against oxidative and environmental stresses. Although studies of thiol metabolism in the model plant Arabidopsis thaliana (thale cress) have expanded our understanding of these dynamic processes, our knowledge of how sulfur is assimilated and metabolized in crop plants, such as soybean (Glycine max), remains limited in comparison. Soybean is a major crop used worldwide for food and animal feed. Although soybeans are protein-rich, they do not contain high levels of the sulfur-containing amino acids, cysteine and methionine. Ultimately, unraveling the fundamental steps and regulation of thiol metabolism in soybean is important for optimizing crop yield and quality. Here we review the pathways from sulfur uptake to glutathione and homoglutathione synthesis in soybean, the potential biotechnology benefits of understanding and modifying these pathways, and how information from the soybean genome may guide the next steps in exploring this biochemical system.
Sulfur is an essential element that must be assimilated by all organisms; however, the metabolic pathways for this task vary significantly, even among individual genera of bacteria, and especially so among eukaryotes. While all organisms require sulfurous amino acids, plants require specialized sulfur-containing metabolites, such as glucosinolates and allylsulfur compounds, for protection from herbivory and microbial infection; and the synthesis of specialized peptides (i.e., glutathione and phytochelatins) for protection against reactive oxygen species and exposure to transition metals, such as cadmium. In order to provide the complex array of sulfur-containing metabolites essential to plant viability, flux through the sulfur assimilatory pathway must be tightly regulated by controlling enzymatic activity. The X-ray crystal structures of several primary sulfur assimilatory enzymes, complemented by kinetics, have revealed mechanisms of enzymatic regulation (i.e., via redox state and protein-protein interaction) in these biosynthetic pathways, in addition to the chemical mechanisms of catalysis. This review summarizes the state of our structural knowledge of primary and secondary sulfur assimilatory enzymes from plants.
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