Serine Acetyltransferase from Escherichia coli Is a Dimer of Trimers

Hindson, V. John; Moody, P.C.E.; Rowe, Arthur J.; Shaw, William V.

doi:10.1074/jbc.275.1.461

Cited by 53 publications

(50 citation statements)

References 23 publications

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“…For this reason, the difference in ␤-sheet strands 4 -6 of AF452452.1 in comparison with GmSATc and other known SATs (supplemental data 6) seems to be responsible for the unique trimeric organization of AF452452.1 and the difference in stoichiometry of the AF452452.1-GmOAS-TLc complex to bacterial and plant CSCs (Figs. 1, 3, and 4) (1,6,12,24,25,29). The altered ␤-sheet domain of AF452452.1 is not expected to be directly involved in the dimerization of SAT trimers or interaction of SAT with OAS-TL (6,11,29).…”

Section: Discussionmentioning

confidence: 99%

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Wirtz

Birke

Heeg

et al. 2010

Journal of Biological Chemistry

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Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (⌬G ‫؍‬ ؊33 kcal mol ؊1 ) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K D ‫؍‬ 30 nM) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K D ) and the regulation of cysteine sensitivity of SAT within the CSC.Cysteine biosynthesis in plants and bacteria is catalyzed by a two-step process. Serine acetyltransferase (SAT 2 ; EC 2.3.1.30) activates serine by transfer of the acetyl moiety from acetyl coenzyme A to form O-acetylserine (OAS). Then OAS accepts sulfide by catalysis of OAS (thiol)-lyase (OAS-TL; EC 2.5.1.47). This fixation of free sulfide from assimilatory sulfate reduction or external sulfide sources is the exclusive entry of reduced sulfur into cellular metabolism. SAT and OAS-TL form the hetero-oligomeric cysteine synthase complex (CSC). In enterobacteria and plants, the interaction of SAT and OAS-TL is stabilized by the presence of sulfide, although the addition of OAS dissociates the two enzymes (1, 2). Plant and bacterial OAS-TLs are dimers that are catalytically inactive in the CSC but become fully active upon dissociation of the complex by OAS (1, 3). However, these properties do not seem to relate to metabolic regulation of cysteine synthesis in enterobacteria. In Escherichia coli, regulation of cysteine synthesis is mainly achieved by control of the cysteine regulon that includes OAS-TL and the genes encoding for proteins catalyzing sulfate uptake and reduction but not bacterial SAT. Bacterial SAT is constitutively expressed but strongly inhibited by cysteine (K I ϭ 1.1 M cysteine). In the presence of cysteine, SAT of E. coli...

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Section: Discussionmentioning

confidence: 99%

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Wirtz

Birke

Heeg

et al. 2010

Journal of Biological Chemistry

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“…However, we note that the CysE structure differs markedly from CdiA-CT EC536 . The CysE C-terminal domain forms a β-helix that terminates in a flexible tail (25). Further, the β-helical domain mediates trimerization, and two CysE trimers interact to form a larger homohexameric complex (25).…”

Section: Discussionmentioning

confidence: 99%

“…The CysE C-terminal domain forms a β-helix that terminates in a flexible tail (25). Further, the β-helical domain mediates trimerization, and two CysE trimers interact to form a larger homohexameric complex (25). If the β-helical domains of CysE interact with CysK, then the contacts are likely to be distinct from those observed with the CdiA-CT EC536 α-helical bundle.…”

Section: Discussionmentioning

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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“…1A) and OASS-A (Fig. 1B) has been determined from different species, including Haemophilus influenzae (9,26,27), E. coli (28,29), Salmonella typhimurium (30 -32), and Arabidopsis thaliana (6,10), the three-dimensional structure of the CS complex has not yet been solved. However, it is well known that the C terminus of SAT (Fig.…”

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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 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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Serine Acetyltransferase from Escherichia coli Is a Dimer of Trimers

Cited by 53 publications

References 23 publications

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

Unraveling the essential role of CysK in CDI toxin activation

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

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