Structure and function of the hetero-oligomeric cysteine synthase complex in plants

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.

FIGURE 1.

Quaternary structure of the AtCSCm and its subunits. A, AtOAS-TLm, AtSATm, and AtCSCm were purified independently and resolved by SEC as described under “Experimental Procedures.” Gray vertical lines represent elution volume of proteins used for calibration (1, thyroglobulin, 669 kDa; 2, ferritin, 440 kDa; 3, catalase, 232 kDa; 4, β-amylase, 200 kDa; 5, aldolase, 158 kDa; 6, alcohol-dehydrogenase, 150 kDa; 7, BSA 66 kDa). B, molecular weights of CSC subunits were independently confirmed by AUC. C, association kinetic of the AtCSCm was analyzed by ITC. The release of heat by subsequent injection of AtOAS-TLm dimer to a AtSATm hexamer is plotted against the molar ratio of AtOAS-TLm dimer to AtSATm hexamer. The solid line represents a fit to data using the one set of equal binding sites model.

FIGURE 2.

Identification of SAT-SAT interaction domains using peptide arrays. A and B, custom-made SAT peptide arrays were probed independently with [³⁵S]Met-labeled AtSATm. In both cases, signal stretches corresponding to part of helix α4 of AtSATm were evident (MNY-⁹²LFDLFSGVLQGN¹⁰³). C, homology modeling of AtSATm to HiSAT revealed that helix α4 shown in space-filling representation is in the same position as the corresponding helix of HiSAT. Helix α4 of HiSAT is known to be involved in the dimerization of bacterial SAT trimers by crystallographic studies. D, side view on α and δ monomer of SAT hexamer that interact via helix α4. The β, χ, ϵ, and γ monomers of the HiSAT hexamer are hidden for clarity.

FIGURE 3.

Quaternary structure of the AtCSCc and its subunits. A, recombinant AtSATc hexamer (0.5 nmol) was purified and resolved by AUC. B, association kinetics of the AtCSCc was analyzed by ITC. The release of heat by subsequent injection of AtOAS-TLc dimer to an AtSATc hexamer is plotted against the molar ratio of AtOAS-TLc dimer to AtSATc hexamer. The solid line represents a fit to the data using the one set of equal binding sites model. C, soluble leaf proteins of 6-week-old Arabidopsis plants were resolved by SEC in the absence or presence of 10 mm OAS to determine the molecular weight of cytosolic CSC and free AtSATc from Arabidopsis, respectively. Equal amounts of fractions (1 ml) corresponding to retention volume 55 to 73 ml were subjected to immunological detection of AtSATc using a specific antiserum.

FIGURE 4.

Quaternary structure of the GmSATc. A, recombinant GmSATc hexamer (0.5 nmol) was resolved by SEC. B, molecular weight of the GmSATC hexamer (1.6 nmol) was confirmed by AUC. C, GmSATc (1.3 nmol) and cytosolic OAS-TL from Arabidopsis (15.5 nmol AtOAS-TLc) were mixed and subjected to AUC. All experiments were performed as described under “Experimental Procedures.”

FIGURE 5.

C-terminal deletion of AtSATm affects enzymatic activity and inhibition by cysteine. A, full-length AtSATm and the C-terminally truncated proteins SATΔC10 and SATΔC15 were purified twice and analyzed for their maximal enzymatic activity (n = 8). B, feedback inhibition of SAT to cysteine was tested. Asterisks indicate significant differences between AtSATm and the truncated SAT proteins (p < 0.05). Statistical analysis was performed with the Student's t test implemented in SigmaStat 3.0 (SPSS).

FIGURE 6.

Cysteine inhibition of plant and bacterial SATs within the CSC. A–C, recombinant EcSAT (A), AtSATc (B), and AtSATm (C) were purified and tested for inhibition of SAT activity by cysteine in free or CSC associated state (n = 4). CSC formation was achieved by co-expression of SAT with its native interaction partner in E. coli followed by histidine-affinity purification of the spontaneously formed CSC. Cysteine concentration was varied between 0 and 0.005 mm for inhibition of bacterial SAT, whereas the concentration of cysteine was increased up to 0.8 mm to efficiently inhibit plant SATs. For comparison, activity of plant and bacterial SAT is shown as percentage of noninhibited free or complex associated SAT.