A Mechanistic Model of the Cysteine Synthase Complex

Feldman‐Salit, Anna; Wirtz, Markus; Hell, Rüdiger; Wade, Rebecca C.

doi:10.1016/j.jmb.2008.08.075

Cited by 72 publications

(73 citation statements)

References 60 publications

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“…This composition supports findings of a recent modeling and docking study that identified a 2:1 ratio of AtOAS-TLm dimer to AtSAT hexamer as the energetically and geometrically most likely composition (24). In this model, one AtSATm trimer binds one AtOAS-TLm dimer.…”

Section: Discussionsupporting

confidence: 90%

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Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Wirtz

Birke

Heeg

et al. 2010

Journal of Biological Chemistry

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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 (⌬G ‫؍‬ ؊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.Cysteine biosynthesis in plants and bacteria is catalyzed by a two-step process. Serine acetyltransferase (SAT 2 ; EC 2.3.1.30) activates serine by transfer of the acetyl moiety from acetyl coenzyme A to form O-acetylserine (OAS). Then OAS accepts sulfide by catalysis of OAS (thiol)-lyase (OAS-TL; EC 2.5.1.47). This fixation of free sulfide from assimilatory sulfate reduction or external sulfide sources is the exclusive entry of reduced sulfur into cellular metabolism. SAT and OAS-TL form the hetero-oligomeric cysteine synthase complex (CSC). In enterobacteria and plants, the interaction of SAT and OAS-TL is stabilized by the presence of sulfide, although the addition of OAS dissociates the two enzymes (1, 2). Plant and bacterial OAS-TLs are dimers that are catalytically inactive in the CSC but become fully active upon dissociation of the complex by OAS (1, 3). However, these properties do not seem to relate to metabolic regulation of cysteine synthesis in enterobacteria. In Escherichia coli, regulation of cysteine synthesis is mainly achieved by control of the cysteine regulon that includes OAS-TL and the genes encoding for proteins catalyzing sulfate uptake and reduction but not bacterial SAT. Bacterial SAT is constitutively expressed but strongly inhibited by cysteine (K I ϭ 1.1 M cysteine). In the presence of cysteine, SAT of E. coli...

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Section: Discussionsupporting

confidence: 90%

“…The peptide stretch identified corresponded to part of the helix ␣4 in the N-terminal AtSATm domain (supplemental data 6). Homology modeling of AtSATm to EcSAT (24) suggests that this ␣-helix is in the same position as the corresponding helix of EcSAT (Fig. 2, C and D).…”

Section: Assay Was Repeated Twice With Freshly Prepared [mentioning

confidence: 87%

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Wirtz

Birke

Heeg

et al. 2010

Journal of Biological Chemistry

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“…Taken together, the van't Hoff analysis and data at high ionic strength suggest that formation of CS involves a desolvation step that drives the encounter between OASS and SAT, whose interaction is mainly stabilized by electrostatic bonds. This finding supports the results of a recent study on A. thaliana CS where the complex generated by docking simulations is mainly stabilized by electrostatic interactions (36). The knowledge of the solvent conditions that stabilize CS can be exploited for the rational optimization of complex crystallization, an elusive goal so far.…”

Section: K Dsupporting

confidence: 88%

“…1C) plays a major role in complex formation (9,21,(33)(34)(35), penetrating inside the active site of OASS and binding at the ␣-carboxylate subsite of the substrate OAS. Simulations of complex formation suggested a role for electrostatic interactions and led to a proposed model for the structure of CS (36). This study and experiments carried out using a variety of different techniques (8,21,36) indicate a 2:1 stoichiometry for the CS complex, with two OASS dimers binding to one SAT hexamer.…”

mentioning

confidence: 71%

“…Simulations of complex formation suggested a role for electrostatic interactions and led to a proposed model for the structure of CS (36). This study and experiments carried out using a variety of different techniques (8,21,36) indicate a 2:1 stoichiometry for the CS complex, with two OASS dimers binding to one SAT hexamer. Binding of SAT to OASS induces large increases in the fluorescence emission of the cofactor, thus allowing the process to be followed (8).…”

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confidence: 71%

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A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

Salsi

Campanini

Bettati

et al. 2010

Journal of Biological Chemistry

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The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

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Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Benoni

Bei

Paredi³

et al. 2017

FEBS Letters

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In bacteria and plants, serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase-A sulfhydrylase (CysK) collaborate to synthesize L-Cys from L-Ser. CysE and CysK bind one another with high affinity to form the cysteine synthase complex (CSC). We demonstrate that bacterial CysE is activated when bound to CysK. CysE activation results from the release of substrate inhibition, with the K i for L-Ser increasing from 4 mM for free CysE to 16 mM for the CSC. Feedback inhibition of CysE by L-Cys is also relieved in the bacterial CSC. These findings suggest that the CysE active site is allosterically altered by CysK to alleviate substrate and feedback inhibition in the context of the CSC. Author contributions BC, SB, and AM conceived and supervised the study; RB, ODB, GP, and NF performed experiments; CSH provided the expression vectors; BC, RB, and ODB analyzed the data; BC prepared the original draft; BC, SB, CSH, and AM reviewed and edited the manuscript. Supporting informationAdditional Supporting Information may be found online in the supporting information tab for this article. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptPlants and bacteria share a common two-reaction pathway for the synthesis of L-cysteine (LCys) from L-serine (L-Ser; Fig. 1). Serine acetyltransferase (CysE) catalyzes an acyl transfer from acetyl-CoA to L-Ser using a random-order kinetic mechanism [1]. The second reaction is catalyzed by O-acetylserine sulfhydrylase-A (CysK), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that displaces the acetoxy group from O-acetylserine with bisulfide to yield L-Cys [2][3][4][5][6][7][8]. Many bacteria also encode O-acetylserine sulfhydrylase-B (CysM) [9,10] that is thought to play an important role in L-Cys biosynthesis under stress conditions [11].Kredich et al. [2,12] first discovered that CysE and CysK from Salmonella Typhimurium bind to one another with high affinity, and they called this assembly the cysteine synthase complex (CSC; Fig. 1). The CysE-CysK interaction is highly conserved across species, and the plant enzymes also form a high-affinity CSC. Although there is no experimentally solved structure available for the CSC, biochemical and spectroscopic approaches revealed that the C-terminal tail of CysE inserts into the CysK active site to anchor the interaction. CysE proteins that lack C-terminal residues are unable to bind CysK [13][14][15], and CSC formation is disrupted by millimolar O-acetylserine, which competes with CysE for binding to the CysK active site [12,16,17]. These findings are supported by crystal structures of CysE Cterminal peptides bound in the active site of CysK. These structures show that the C-terminal Ile residue of CysE engages in the same specific interactions with the active site as Oacetylserine substrate [18,19]. The stoichiometry of CysE to CysK has been determined to be 3:2 for CSCs from S. Typhimurium and Haemophilus influenzae. Because CysK forms homodimers and CysE exists as a dimer of trimers [20,21],...

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A Mechanistic Model of the Cysteine Synthase Complex

Cited by 72 publications

References 60 publications

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

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