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

Cysteine is very susceptible to reactive oxygen species. In response; posttranslational thiol modifications such as reversible disulfide bond formation have arisen as protective mechanisms against undesired in vivo cysteine oxidation. In Gram-negative bacteria a major defense mechanism against cysteine overoxidation is the formation of mixed protein disulfides with low molecular weight thiols such as glutathione and glutathionylspermidine. In this review we discuss some of the mechanistic aspects of glutathionylspermidine in prokaryotes and extend its potential use to eukaryotes in proteomics and biochemical applications through an example with tissue transglutaminase and its S-glutathionylation. OPEN ACCESS

Cellular redox conditions affect Gsp amidase activity in Escherichia coli. Guided by the structure and catalytic mechanism of the amidase, we designed and synthesized an acyloxymethyl ketone-based activity probe containing a biotin handle. This probe was used to monitor Gsp amidase activity in E. coli lysates that had been subjected to oxidative or methylglyoxal-induced stress.