Isolation of serine acetyltransferase complexed with cysteine synthase from Allium tuberosum.

Nakamura, Katsuhito; Tamura, Goro

doi:10.1271/bbb1961.54.649

Cited by 27 publications

(6 citation statements)

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“…In plants, SATase is usually associated with cysteine synthase to form a complex [5–9]. In this study, we have not investigated whether the recombinant plant SATases can form a complex with E. coli cysteine synthase.…”

Section: Discussionmentioning

confidence: 99%

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Determination of the sites required for the allosteric inhibition of serine acetyltransferase by L‐cysteine in plants

Inoue

Noji

Saito

1999

European Journal of Biochemistry

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Serine acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetylserine from l-Ser and acetylCoA in plants and bacteria. In plants, two types of SATase have been described. One is allosterically inhibited by l-Cys, and the second is not sensitive to l-Cys inhibition. However, the allosteric site in SATase has not been identified. To understand better the mechanism of l-Cys inhibition of plant SATases, we constructed several chimeric SATase enzymes from watermelon SATase (WaSATase) (sensitive type) and Arabidopsis SAT-p (insensitive type). These enzymes were expressed in Escherichia coli, and inhibition of the mutated SATase activity by l-Cys was analyzed. Mutated WaSATase, in which Met280 was changed to Ile, was no longer inhibited by l-Cys. Analysis of the inhibition the chimeric enzymes indicated that the C-terminal region of WaSATase from Pro276 to Phe285, in which five amino acids are different from those of SAT-p, was responsible for the determination of the sensitivity to l-Cys. In particular, Gly277 in the C-terminal region of WaSATase was primarily responsible for the l-Cys inhibition. The N-terminal half of the protein, which does not contain the catalytic domain, was also important for the sensitivity to l-Cys. These results indicate that the sensitivity of SATase to l-Cys is due to the N-terminal and C-terminal regions rather than to the catalytic domain.

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Section: Discussionmentioning

confidence: 99%

“…O ‐acetylserine then reacts with sulfide to yield l ‐Cys through the action of cysteine synthase [ O ‐acetylserine(thiol)‐lyase; EC 4.2.99.28]. It is known that SATase can be multimeric, and is able to form a complex with cysteine synthase [5–9]. SATase activity in bacteria is inhibited by micromolar concentrations of l ‐Cys [10].…”

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confidence: 99%

Determination of the sites required for the allosteric inhibition of serine acetyltransferase by L‐cysteine in plants

Inoue

Noji

Saito

1999

European Journal of Biochemistry

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“…Moreover, because the CysK active site is physically occluded by CysE, the complex actually belies its name and inefficiently produces l‐ Cys. Studies conducted with the plant complex suggest that the CysE–CysK interaction serves primarily to modulate substrate flux through CysE, thereby tuning the rate of l‐ Cys biosynthesis . CysK binding not only promotes the activity of CysE but also protects the enzyme from cold‐inactivation and proteolytic destruction .…”

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confidence: 99%

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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“…By contrast, the regulatory role of plant CSC is much better characterized. In plants, CysK is present in large excess over CysE (about 300 times more abundant in spinach chloroplasts) [15, 64, 65], and CysE is activated upon complex formation [2, 19, 25, 28]. Very recent data on Glycine max CysE indicate a 20-fold increase in catalytic efficiency upon binding to CysK.…”

Section: The Regulatory Function Of Cysk/cyse Interaction In Plantsmentioning

confidence: 99%

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein

Campanini

Benoni

Bettati

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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O -acetylserine sulfhydrylase A (CysK) is the pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This “cysteine synthase” complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40 years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional “moonlighting” activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode C. elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site.

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Isolation of serine acetyltransferase complexed with cysteine synthase from Allium tuberosum.

Cited by 27 publications

References 2 publications

Determination of the sites required for the allosteric inhibition of serine acetyltransferase by L‐cysteine in plants

Determination of the sites required for the allosteric inhibition of serine acetyltransferase by L‐cysteine in plants

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

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein

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