Geoffrey E. Ravilious

Sensing sulfur conditions: simple to complex protein regulatory mechanisms in plant thiol metabolism.

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 natures 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.

From sulfur to homoglutathione: thiol metabolism in soybean.

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.

Structural basis and evolution of redox regulation in plant adenosine-5′-phosphosulfate kinase

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, Mg2+, 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 kcat/Km and a 15-fold increase in Ki 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.

Structural biology of plant sulfur metabolism: From assimilation to biosynthesis

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.

Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation

Background: ATP sulfurylase catalyzes the energetically unfavorable formation of adenosine 5′-phosphosulfate in plant sulfur assimilation. Results: Structural and kinetic analyses identifies key active site residues. Conclusion: A reaction mechanism involving distortion of nucleotide conformation and stabilizing interactions is proposed. Significance: These results provide the first molecular insights on a plant ATP sulfurylase and the committed step of plant sulfur assimilation. Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5′-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3′-phosphoadenosine 5′-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway.

Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein

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. Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP.

Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5'-Phosphosulfate Kinase

Background: Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the synthesis of phosphoadenosine 5′-phosphosulfate, but how APSK coordinates binding of phosphonucleotides is unclear. Results: Using calorimetry, crystallography, and mutagenesis, this study provides new insight on nucleotide binding in APSK. Conclusion: The P-loop and a critical aspartate integrate dynamic structural and nucleotide recognition features. Significance: These results suggest how structural changes guide the order of nucleotide addition for catalysis. Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3′-phosphate 5′-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

Kinetic mechanism of the dimeric ATP sulfurylase from plants.

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.

Redox-linked Gating of Nucleotide Binding by the N-terminal Domain of Adenosine 5′-Phosphosulfate Kinase

Background: Adenosine 5′-phosphosulfate kinase (APSK) in plants contains a regulatory disulfide bond. Results: Analysis of oxidized APSK reveals altered nucleotide binding compared with the reduced enzyme. Conclusion: The N-terminal domain is responsible for redox-linked structural changes that regulate APSK activity. Significance: This work provides a molecular basis for understanding reciprocal regulation at a branch point in plant sulfur metabolism. Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine 5′-phosphosulfate (APS) to 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Crystallographic studies of APSK from Arabidopsis thaliana revealed the presence of a regulatory intersubunit disulfide bond (Cys86–Cys119). The reduced enzyme displayed improved catalytic efficiency and decreased effectiveness of substrate inhibition by APS compared with the oxidized form. Here we examine the effect of disulfide formation and the role of the N-terminal domain on nucleotide binding using isothermal titration calorimetry (ITC) and steady-state kinetics. Formation of the disulfide bond in A. thaliana APSK (AtAPSK) inverts the binding affinities at the ATP/ADP and APS/PAPS sites from those observed in the reduced enzyme, consistent with initial binding of APS as inhibitory, and suggests a role for the N-terminal domain in guiding nucleotide binding order. To test this, an N-terminal truncation variant (AtAPSKΔ96) was generated. The resulting protein was completely insensitive to substrate inhibition by APS. ITC analysis of AtAPSKΔ96 showed decreased affinity for APS binding, although the N-terminal domain does not directly interact with this ligand. Moreover, AtAPSKΔ96 displayed reduced affinity for ADP, which corresponds to a loss of substrate inhibition by formation of an E·ADP·APS dead end complex. Examination of the AtAPSK crystal structure suggested Arg93 as important for positioning of the N-terminal domain. ITC and kinetic analysis of the R93A mutant also showed a complete loss of substrate inhibition and altered nucleotide binding affinities, which mimics the effect of the N-terminal deletion. These results show how thiol-linked changes in AtAPSK alter the energetics of binding equilibria to control its activity.

An autoinhibitory conformation of the Bacillus subtilis spore coat protein SpoIVA prevents its premature ATP‐independent aggregation

Spores of Bacillus subtilis are dormant cell types that are formed when the bacterium encounters starvation conditions. Spores are encased in a shell, termed the coat, which is composed of approximately seventy different proteins and protects the spores genetic material from environmental insults. The structural component of the basement layer of the coat is an exceptional cytoskeletal protein, termed SpoIVA, which binds and hydrolyzes ATP. ATP hydrolysis is utilized to drive a conformational change in SpoIVA that leads to its irreversible self-assembly into a static polymer in vitro. Here, we characterize the middle domain of SpoIVA, the predicted secondary structure of which resembles the chemotaxis protein CheX but, unlike CheX, does not harbor residues required for phosphatase activity. Disruptions in this domain did not abolish ATP hydrolysis, but resulted in mislocalization of the protein and reduction in sporulation efficiency in vivo. In vitro, disruptions in this domain prevented the ATP hydrolysis-driven conformational change in SpoIVA required for polymerization and led to the aggregation of SpoIVA into particles that did not form filaments. We propose a model in which SpoIVA initially assumes a conformation in which it inhibits its own aggregation into particles, and that ATP hydrolysis remodels the protein so that it assumes a polymerization-competent conformation.