On the interaction site of serine acetyltransferase in the cysteine synthase complex from Escherichia coli

Zhao, Chunhui; Moriga, Yudai; Feng, Bin; Kumada, Yoichi; Imanaka, Hiroyuki; Imamura, Koreyoshi; Nakanishi, Koji

doi:10.1016/j.bbrc.2006.01.054

Cited by 44 publications

(68 citation statements)

References 23 publications

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“…It seems plausible that the presence of chloride and bisulfide mainly affects k 4 , whose value, however, cannot be determined with accuracy due to large errors associated with extrapolation to zero ligand concentration. In agreement with this observation, surface plasmon resonance experiments revealed that low affinity mutants of E. coli SAT exhibit an invariant value of k on but a larger value of k off (44). However, previous studies (44) showed that k off values in the presence of bisulfide are of the same order of magnitude as those in its absence.…”

Section: K Dsupporting

confidence: 61%

“…In agreement with this observation, surface plasmon resonance experiments revealed that low affinity mutants of E. coli SAT exhibit an invariant value of k on but a larger value of k off (44). However, previous studies (44) showed that k off values in the presence of bisulfide are of the same order of magnitude as those in its absence. It should be noted, however, that by surface plasmon resonance, only an overall k off value can be calculated, i.e.…”

Section: K Dsupporting

confidence: 61%

“…The CS bienzyme complex is stabilized by bisulfide (21,44), but the meaning of this effect is poorly understood. The pK a value for the H 2 S to SH Ϫ ionization is 7, and experiments were carried out at pH 8 where H 2 S is more than 90% dissociated to bisulfide (43,45).…”

Section: Effect Of Cysteine and Bisulfide On The Kinetics Of Formatiomentioning

confidence: 99%

“…Bisulfide is the product of cysteine desulfuration by intracellular desulfurases and could thus inhibit the buildup of the toxic molecule cysteine above a given concentration (30). Furthermore, a stabilizing effect of bisulfide on CS is reported (21,44). The effect of chloride and bisulfide on the kinetics of complex formation is different, with chloride giving a 16-fold decrease of the second order kinetic constant k 3 /K d and bisulfide giving almost no effect.…”

Section: K Dmentioning

confidence: 99%

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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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Section: K Dsupporting

confidence: 61%

Section: K Dsupporting

confidence: 61%

Section: Effect Of Cysteine and Bisulfide On The Kinetics Of Formatiomentioning

confidence: 99%

Section: K Dmentioning

confidence: 99%

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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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“…In bacteria and plants, CysK is found in the "cysteine synthase" complex together with CysE-the serine O-acetyltransferase responsible for the penultimate step of cysteine synthesis (10). Formation of the cysteine synthase complex requires the C-terminal tail of CysE, which inserts into the CysK active site (11). The C-terminal Ile Significance Contact-dependent growth inhibition (CDI) systems produce toxins that inhibit competing bacteria and immunity proteins that protect against self-inhibition.…”

mentioning

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

et al. 2016

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O‐acetylserine sulfhydrylase (OASS), the enzyme catalysing the last step of cysteine biosynthesis in bacteria, is involved in antibiotic resistance and biofilm formation. Since mammals lack OASS, it is a potential target for antimicrobials. However, a limited number of inhibitors has been developed and crystallized in complex with OASS. STD‐NMR was applied to study the interaction of the inhibitory pentapeptide MNYDI with the CysK and CysM OASS isozymes from Salmonella Typhimurium. Results are in excellent agreement with docking and SAR studies and confirm the important role played by the C‐terminal Ile5 and the arylic moiety at P3 in dictating affinity.

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On the interaction site of serine acetyltransferase in the cysteine synthase complex from Escherichia coli

Cited by 44 publications

References 23 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

Unraveling the essential role of CysK in CDI toxin activation

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

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