Protein S-Thiolation by Glutathionylspermidine (Gsp)

Abstract

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

Polyamine catabolism and oxidative damage

“…Glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the formation and removal of an amide bond between GSH and Spd to govern Gsp abundance (27). The amidase domain of GspSA is sensitive to inactivation by oxidation, resulting in Gspmodified proteins, including Gsp disulfides and protein thiols (28). These data are consistent with the hypothesis that the formation/ hydrolysis of Gsp represents an oxidative stress mechanism that contributes to the maintenance of redox homeostasis in E. coli.…”

In Vivo Tagging and Characterization of S‐Glutathionylated Proteins by a Chemoenzymatic Method

“…The enzyme GspS catalyzes the amide bond formation between GSH and spermidine to generate glutathionylspermidine (Gsp; Figure 1 in the Supporting Information), a unique GSH derivative found only in a few Gram‐negative bacteria and protozoa 8. We previously discovered that Gsp behaves similarly to GSH in forming disulfide bonds with cysteine residues of proteins in vivo 9. Consequently, if the enzyme GspS is expressed in mammalian cells and generates Gsp, the mixed‐disulfide bond formation between Gsp and protein cysteine residues would be akin to in situ protein glutathionylation.…”

An Acyloxymethyl Ketone‐Based Probe to Monitor the Activity of Glutathionylspermidine Amidase in Escherichia coli

“…[9] The catalytic triad of GspA, Cys59, His131, and Glu147, which is completely conserved in CHAP-domain proteins, forms a hydrogenbond network that enhances the nucleophilicity of Cys59. [7] Previous results indicated that the inactivation of amidase activity increases Gsp level in E. coli, which then protects against harmful oxidants as follows. [7] Oxidants are consumed when Gsp-Gsp disulfides (or other mixed disulfides) are formed.…”

“…[7] Previous results indicated that the inactivation of amidase activity increases Gsp level in E. coli, which then protects against harmful oxidants as follows. [7] Oxidants are consumed when Gsp-Gsp disulfides (or other mixed disulfides) are formed. Gsp also prevents the oxidation of protein cysteine thiols by Gspprotein cysteine disulfide formation.…”

“…[6] We previously reported that the activity of GspA was selectively inactivated by H 2 O 2 , whereas H 2 O 2 hardly affected Gsp synthetase activity. [7] In addition, the GspA activity in GspSA is negatively regulated by the synthetase activity, although this depends on the combination of synthetase substrates/analogues. [8] The aforementioned observations are indications of a rather complicated regulation of GspSA activity in response to environmental pressures.…”