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

Abstract

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...

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

“…Binding of APS was weaker than ATP and was insensitive to Mg 2ϩ (Fig. 2C, Table 2), which is consistent with the location of the ion in the ATP/ADP, not APS/ PAPS, binding site of AtAPSK (25).…”

“…The standard experimental buffer condition was 25 mM HEPES, pH 7.5, 200 mM KCl, 5% (v/v) glycerol, and either 1 mM Tris(2-carboxyethyl)phosphine or 5 mM ␤-mercaptoethanol (␤ME), unless stated otherwise. Either reducing agent prevented formation of intermonomer disulfide bonds (25), as judged by nonreducing SDS-PAGE of samples taken before and after ITC titrations.…”

“…Protein overexpression in E. coli BL21(DE3) and purification by nickel affinity and gel-filtration chromatographies were described previously (25), with some modifications. For cell lysis and nickel affinity chromatography, all buffers were supplemented with 5 mM ␤ME.…”

“…Enzyme Assays-All steady-state kinetic analysis was performed as previously described using an enzyme-coupled spectrophotometric assay (25).…”

“…1B) and P. chrysogenum, the APSK domain from human PAPS synthetase, and the bifunctional ATP sulfurylase-APSK from Aquifex aeolicus and Thiobacillus denitrificans (25)(26)(27)(28)(29)(30) show that the overall fold of APSK is highly conserved across a variety of organisms. Two nucleotide-binding sites (i.e.…”