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

Abstract

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],...

Activation of an anti-bacterial toxin by the biosynthetic enzyme CysK: mechanism of binding, interaction specificity and competition with cysteine synthase

“…1A ). This effect is very similar to that observed for the cysteine synthase complexes of Haemophilus influenzae 38 and E. coli 55 . Moreover, the fluorescence spectrum of EcCysK:CdiA-CT exhibited a blue-shift in the emission maximum from 505 to 498 nm compared to free EcCysK (Fig.…”

“…This value is in agreement with the EcCysK:CdiA-CT binding constant calculated from surface plasmon resonance data 57 . Moreover, EcCysE inhibits EcCysK activity with a K i of 6.2 ± 0.7 nM 55 , indicating that the toxin and EcCysE bind to EcCysK with similar affinities.…”

“…We first used an indirect approach to test whether CdiA-CT interferes with the assembly of cysteine synthase complexes. Because EcCysE activity is stimulated when bound to EcCysK 55 , we titrated the CS complex with CdiA-CT and measured serine acetyltransferase activity. The maximal rate of serine acetylation was obtained with 28 nM EcCysE and 19 nM EcCysK, conditions in which the two proteins are at stoichiometric amounts based on the 3:2 CysE:CysK stoichiometry of the cysteine synthase complex.…”

“…Briefly, O -acetylation of 20 mM L-Ser was monitored by measuring the absorption at 232 nm of the thioester bond (ε 232 = 4,440 M −1 cm −1 ), while varying acetyl-CoA concentrations. At a fixed 0.25 mM acetyl-CoA concentration, EcCysE activity increases as a function of EcCysK concentration 55 . Displacement of EcCysE from the cysteine synthase complex by CdiA-CT was monitored using 28 nM CysE (4.7 nM hexamer) in the presence of 19 nM CysK (9.5 nM dimer).…”

Refining the structure−activity relationships of 2-phenylcyclopropane carboxylic acids as inhibitors of O-acetylserine sulfhydrylase isoforms

“…OASS-A is highly expressed at basal levels and together with SAT forms the cysteine synthase complex, whose function is still controversial 1–4 , 9 . Interestingly, SAT physiologically inhibits OASS activity through its carboxy-terminal portion by competing with the natural substrate OAS for binding to the active site.…”