Evidence for a Glutathionyl-Enzyme Intermediate in the Amidase Activity of the Bifunctional Glutathionylspermidine Synthetase/Amidase from <i>Escherichia coli</i>

Ch, Lin; Ds, Kwon; Jm, Bollinger; Ct, Walsh

doi:10.1021/bi9714464

Cited by 30 publications

(31 citation statements)

References 17 publications

(40 reference statements)

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“…TcTryS also has an opposing amidase activity, which is about 1% of the synthetase activity when assayed at pH 8.0 near the optima for the forward and reverse reactions. This is consistent with our finding that TcTryS has an N-terminal domain containing a conserved Cys (at position 51) previously shown to be essential for amidase activity in both E. coli (35) and C. fasciculata (27). However, the amidase activity of TcTryS with glutathionylspermidine is two orders of magnitude lower than that observed for GspS from C. fasciculata, when assayed under identical conditions (Table II).…”

Section: Discussionsupporting

confidence: 92%

“…Significant homology was found between TcTryS and EcGspS in the N-terminal region, which is responsible for the glutathionylspermidine amidase activity of the E. coli protein (28,34). Moreover, a cysteine residue, corresponding to Cys-59 of EcGspS and Cys-79 Cf GspS that have been shown by site-directed mutagenesis to be essential for amidase activity (27,35), is present in TcTryS, suggesting that this enzyme may also possess amidase activity (see below).…”

Section: Resultsmentioning

confidence: 99%

“…A similar activity has not been reported for other trypanosomatids. The mechanism by which the opposing synthetase and amidase functions are regulated merits further investigation, particularly whether binding of substrate(s) of the synthetase domain enhances amidase activity as reported for E. coli GspS (35).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

A Single Enzyme Catalyses Formation of Trypanothione from Glutathione and Spermidine in Trypanosoma cruzi

Oza¹,

Tétaud²,

Ariyanayagam

et al. 2002

Journal of Biological Chemistry

119

100

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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A Single Enzyme Catalyses Formation of Trypanothione from Glutathione and Spermidine in Trypanosoma cruzi

Oza¹,

Tétaud²,

Ariyanayagam

et al. 2002

Journal of Biological Chemistry

119

100

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“…Reagents and Chemicals-The chromogenic tripeptide ␥-Glu-Ala-Gly-p-nitroanilide (␥-EAG-pNA), which was the amidase substrate, was synthesized as reported (10). The thiol-labeling reagent, monobromobimane (mBBr) and (GspS) 2 were purchased from Bachem and Invitrogen, respectively.…”

Section: Methodsmentioning

confidence: 99%

Protein S-Thiolation by Glutathionylspermidine (Gsp)

Chiang

Chen

Pai

et al. 2010

Journal of Biological Chemistry

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Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated by H 2 O 2 oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA and GspSA/ glutaredoxin null mutants to H 2 O 2 support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli.Protein thiols are readily oxidized and reduced to form sulfenates, sulfinates, sulfonates, and intra-and intermolecular disulfides. Most organisms have complex systems that regulate the intracellular redox states of thiols (1, 2). Small thiol-containing biomolecules (e.g. GSH and cysteine, form mixed-disulfides with protein thiols (S-thiolation). These post-translational modifications protect proteins from overoxidation and regulate certain protein functions (3, 4). For example, S-glutathionylation of Escherichia coli methionine synthase, which occurs when E. coli is oxidatively stressed, suppresses the synthase activity, thereby decreasing cellular methionine concentration (5). Because GSH is abundant in most organisms (often existing at 1-10 mM), protein S-glutathionylation (GSH S-thiolation) is considered to be a reversible and universal cellular process. In E. coli, GSH and spermidine (Spd) 4 form N 1

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“…The function of this compound in E. coli is not known, although it has been suggested that it is involved in regulating the levels of the precursors (spermidine and glutathione). More recently, Bollinger et al (23)(24)(25) reported the cloning and characterization of a bifunctional E. coli glutathionylspermidine synthetase/amidase enzyme responsible for both the biosynthesis and degradation of glutathionylspermidine in E. coli.…”

mentioning

confidence: 99%

Cloning and Characterization of the Two Enzymes Responsible for Trypanothione Biosynthesis in Crithidia fasciculata

Tétaud¹,

Manai²,

Barrett³

et al. 1998

Journal of Biological Chemistry

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Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (␥-Glu-Cys-Gly) and spermidine to form trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSS and Cf-TRS share 50% sequence similarity with the Escherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli and Saccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with the Cf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both the Cf-GSS and Cf-TRS genes contained glutathionylspermidine and trypanothione, confirming that these genes encode the functional glutathionylspermidine and trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coli glutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.

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Evidence for a Glutathionyl-Enzyme Intermediate in the Amidase Activity of the Bifunctional Glutathionylspermidine Synthetase/Amidase from Escherichia coli

Cited by 30 publications

References 17 publications

A Single Enzyme Catalyses Formation of Trypanothione from Glutathione and Spermidine in Trypanosoma cruzi

A Single Enzyme Catalyses Formation of Trypanothione from Glutathione and Spermidine in Trypanosoma cruzi

Protein S-Thiolation by Glutathionylspermidine (Gsp)

Cloning and Characterization of the Two Enzymes Responsible for Trypanothione Biosynthesis in Crithidia fasciculata

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