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],...
Cysteine is a building block for many biomolecules that are crucial for living organisms. O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals, catalyzes the last step of cysteine biosynthesis. This enzyme has been deeply investigated because, beside the biosynthesis of cysteine, it exerts a series of ''moonlighting'' activities in bacteria. We have previously reported a series of molecules capable of inhibiting Salmonella typhimurium (S. typhymurium) OASS isoforms at nanomolar concentrations, using a combination of computational and spectroscopic approaches. The cyclopropane-1,2-dicarboxylic acids presented herein provide further insights into the binding mode of small molecules to OASS enzymes. Saturation transfer difference NMR (STD-NMR) was used to characterize the molecule/ enzyme interactions for both OASS-A and B. Most of the compounds induce a several fold increase in fluorescence emission of the pyridoxal 5 0 -phosphate (PLP) coenzyme upon binding to either OASS-A or OASS-B, making these compounds excellent tools for the development of competition-binding experiments.
Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an O-acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, Klebisiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pseudintermedius. The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure−activity relationship studies.
The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.
In ϒ-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of l-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC50 <100 μM were identified via an indirect assay using Ellman’s reagent and then tested on a Gram-negative model organism, with one of them being able to interfere with bacterial growth via SAT inhibition.
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