Structural insight into the interaction of <i>O‐</i>acetylserine sulfhydrylase with competitive, peptidic inhibitors by saturation transfer difference‐<scp>NMR</scp>

Benoni, Roberto; Pertinhez, Thelma A.; Spyrakis, Francesca; Davalli, Silvia; Pellegrino, Sara; Paredi, Gianluca; Pezzotti, Angelica; Bettati, Stefano; Campanini, Barbara; Mozzarelli, Andrea

doi:10.1002/1873-3468.12126

Cited by 11 publications

(7 citation statements)

References 55 publications

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“…By contrast, mutations in the l‐ Cys synthetic pathway interfere with biofilm formation in Vibrio fischeri . l‐ Cys metabolism has also been linked to antibiotic resistance in S. Typhimurium and is actively being explored as a target for novel antimicrobial therapies and the development of antibiotic enhancers . This approach is beginning to produce some encouraging results, particularly in the case of Mycobacterium tuberculosis .…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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 our previous investigations, we carried out a series of saturation transfer difference (STD) NMR measurements, in order to define the interaction between a given ligand and the enzyme 39 . The analysis of the STD-NMR spectra allows mapping the ligand protons directly involved in the interaction with the enzyme 40 .…”

Section: Std-nmr Analysismentioning

confidence: 99%

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation ofO-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

Annunziato¹,

Pieroni²,

Benoni

et al. 2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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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.

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“…For example, cysteine biosynthesis pathway plays an important role in the biofilm formation of some bacteria (17). In Staphylococcus mutans, O-acetylserine sulfatase (CysK) overexpression increases cysteine synthesis, which in turn promotes biofilm formation (31).…”

Section: Discussionmentioning

confidence: 99%

“…Deletions and overexpression of genes which regulate AI-2 also promote biofilm formation (3). The cysteine biosynthesis pathway, an amino acid metabolism pathway of bacteria, and its associated enzymes, substrates, and products are closely related to biofilm (17). In the cysteine biosynthetic pathway, O-acetylserine (thiol)-lyase B (CysM) is expressed not only under anaerobic conditions, but also catalyzes the synthesis of L-cysteine from O-acetylserine sulfhydrylase (OASS) and thiosulfate (18).…”

Section: Introductionmentioning

confidence: 99%

Tylosin Inhibits Streptococcus suis Biofilm Formation by Interacting With the O-acetylserine (thiol)-lyase B CysM

Zhou

Chen

et al. 2022

Front. Vet. Sci.

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Streptococcus suis (S. suis) can decrease its virulence or modify local conditions through biofilm formation, which promotes infection persistence in vivo. Biofilm formation is an important cause of chronic drug-resistant S. suis infection. The aim of this study was to evaluate whether tylosin effectively inhibits S. suis biofilm formation by interacting with O-acetylserine (thiol)-lyase B (CysM), a key enzymatic regulator of cysteine synthesis. Biofilm formation of the mutant (ΔcysM) strain was significantly lower compared to the wild-type ATCC 700794 strain. Tylosin inhibited cysM gene expression, decreased extracellular matrix contents, and reduced cysteine, homocysteine, and S-adenosylmethionine levels, indicating its potential value as an effective inhibitor of S. suis biofilm formation. Furthermore, using biolayer interferometry technology and fourier-transform infrared spectroscopy, we found that tylosin and CysM could be combined directly. Overall, these results provide evidence that tylosin inhibits S. suis biofilm formation by interacting with CysM.

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Structural insight into the interaction of O‐acetylserine sulfhydrylase with competitive, peptidic inhibitors by saturation transfer difference‐NMR

Cited by 11 publications

References 55 publications

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

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

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation ofO-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

Tylosin Inhibits Streptococcus suis Biofilm Formation by Interacting With the O-acetylserine (thiol)-lyase B CysM

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