Interaction of serine acetyltransferase with <i>O</i>‐acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

Campanini, Barbara; Speroni, Francesca; Salsi, Enea; Cook, Paul F.; Roderick, Steven L.; Huang, Bin; Bettati, Stefano; Mozzarelli, Andrea

doi:10.1110/ps.051492805

Cited by 82 publications

(133 citation statements)

References 56 publications

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“…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: 64%

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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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confidence: 64%

“…In Escherichia coli OASS-A also interacts with ATP sulfurylase (19) and in Bacillus subtilis with the repressor CymR (14). OASS-A is known to interact with SAT to form a very tight (K d Х1 nM) complex (8). In plants, CS acts as a sensor for the levels of sulfur inside the cell, and in enterobacteria, CS function has not yet been assessed (20).…”

mentioning

confidence: 99%

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 binding affinity of ligands to OASS was determined by monitoring the increase in fluorescence emission of the PLP coenzyme following excitation at 412 nm 24,32 . Emission spectra were recorded as a function of ligand concentration in a solution containing 0.5-1.0 mM OASS, 100 mM Hepes buffer, pH 7.0, at 20 C. Fluorescence measurements were carried out using a FluoroMax-3 fluorometer (HORIBA), equipped with a thermostatted cell holder, and spectra were corrected for blank contribution.…”

Section: Direct Determination Of Ligand-binding Affinity To Oassmentioning

confidence: 99%

“…One interesting property of most compounds reported here is the high fluorescence quantum yield of the complex with either OASS-A or OASS-B (Table 1). Taking into account that the formation of the complex between the C-terminal decapeptide of SAT and OASS leads to a 4-fold increase in fluorescence emission intensity, likely due to changes in the binding pocket microenvironment and/or shielding from solvent quenching 32 , some of the compounds identified in this work are far more efficient in exerting such an effect. For example, saturating concentrations of compound 19 and compound 20 lead to a 9-and 10-fold increase in the fluorescence emission of the cofactor of the A isoform (Table 1).…”

Section: Fluorescence Properties Of the Oass/compounds Complexesmentioning

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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“…Although CysK and CysM share 58% sequence identity, CysE does not interact stably with the CysM isoenzyme (14). The CdiA-CT EC536 toxin carries a C-terminal Gly-Tyr-Gly-Ile (GYGI) peptide motif that appears to mimic the Gly-Asp-Gly-Ile (GDGI) tail of E. coli CysE.…”

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confidence: 99%

Unraveling the essential role of CysK in CDI toxin activation

Johnson

Beck

Morse

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI + bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT EC536 deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT EC536 binary complex and the neutralized ternary complex of CysK/CdiA-CT/ CdiI EC536 . CdiA-CT EC536 inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT EC536 exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT EC536 thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT EC536 , and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.bacterial competition | structural biology | toxin chaperone | tRNase activity

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Interaction of serine acetyltransferase with O‐acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

Cited by 82 publications

References 56 publications

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

A Two-step Process Controls the Formation of the Bienzyme 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

Unraveling the essential role of CysK in CDI toxin activation

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