Abstract
Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = -33 kcal mol(-1)) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K(D) = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K(D)) and the regulation of cysteine sensitivity of SAT within the CSC.
Highlights
Cysteine biosynthesis in plants and bacteria is catalyzed by a two-step process
Apparent molecular sizes were determined by molecular size exclusion chromatography (SEC) as 72 Ϯ 5 kDa for AtOAS-TLm (n ϭ 7), 208 Ϯ 12 kDa for AtSATm (n ϭ 6), and 343 Ϯ 45 kDa for cysteine synthase complex (CSC) (n ϭ 7) (Fig. 1A)
Based on the calculated molecular weights, these sizes correspond to a dimer of AtOAS-TLm, a hexamer of AtSATm, and a 2:1 ratio of the AtOAS-TLm dimer to the AtSATm hexamer in the CSC
Summary
General Cloning—PCR and cloning of DNA fragments were performed according to Ref. 18. The same strategy was applied for cloning of full-length AtSATc (At5g56760), GmSATc (Glyma16g03080), and histidine-tagged AtOAS-TLc (At4g14880) using the primer combinations p_1366, p_1367, p_1364, p_1365, p_202, and p_203 (supplemental data 1). AtSATc and GmSATc were purified via their affinity to OAS-TL, after immobilization of histidine-tagged AtOAS-TLc on a HiTrapTM Chelating HP column (GE Healthcare) as bait. Elution of mature AtSATc and GmSATc was achieved by dissociation of CSC with 10 mM OAS in 50 mM Tris, pH 7, 0.25 M sodium chloride for size exclusion chromatography or 0.1 M HEPES, pH 7, 0.25 M sodium chloride for analytical ultracentrifugation. Analytical Ultracentrifugation—Centrifugation was performed in 50 mM Tris, pH 8.5, 0.25 M sodium chloride for AtSATm and AtOAS-TLm and in 0.1 M HEPES, pH 7, 0.25 M sodium chloride for AtSATc, GmSATc, and AtOAS-TLc. Protein concentration varied between 0.5 and 5 mg mlϪ1. The antiserum was demonstrated to be specific by comparison of signals determined in crude extracts of wild type and a T-DNA knock-out line of AtSATc (supplemental data 3)
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