Protein S-thiolation by Glutathionylspermidine (Gsp): the role of Escherichia coli Gsp synthetASE/amidase in redox regulation

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.

FIGURE 1.

Detection of GspSSPs in E. coli. A, a schematic diagramming how Gsp S-thiolation of E. coli proteins was detected. ¹⁴C-Labeled spermidine (Spd*) was added into an E. coli culture and was then conjugated with GSH by Gsp synthetase activity in GspSA to form radiolabeled Gsp (Gsp*). Gsp*SSPs were detected by phosphorimaging after SDS-PAGE. The levels of radiolabeled proteins were reduced when extracts were treated with 2-ME. B, SDS-PAGE analysis of E. coli proteins in NR754 (wild type) and HA61002 (ΔgspSA). Left, Coomassie Brilliant Blue-stained proteins. Right, phosphorimaging of radiolabeled proteins.

FIGURE 2.

Gsp accumulation in E. coli after H₂O₂ treatment. The levels of Gsp and GspSA in E. coli were measured after the bacteria had been cultured in M9 minimal medium, treated with H₂O₂, lysed, and treated with mBBr. A, HPLC chromatograms of mBBr-derivatized thiol compounds detected using fluorescence spectroscopy. The arrows indicate the elution position of Gsp. The numbers above the arrows are the amounts (μmol) of Gsp obtained from 1 mg, dry weight, of cells. B, SDS-PAGE of GspSA shows that its expression level was unaffected by H₂O₂.

FIGURE 3.

Effects of H₂O₂ on GspSA amidase and synthetase activities. A, plots of Gsp amidase activity that remained after treatment with four different concentrations of H₂O₂. Activity was measured using the chromogenic substrate γ-EAG-pNA. B, plots of Gsp synthetase activity that remained after treatment with four different concentrations of H₂O₂. At worst, there was only a 10% reduction in synthetase activity compared with that of the negative control (0 m H₂O₂). Synthetase activity was measured as the consumption of NADH by the pyruvate kinase/lactate dehydrogenase couple, which also used ADP generated by Gsp synthetase.

FIGURE 4.

Identification of the Cys⁵⁹ sulfenic acid by x-ray crystallography and chemical modification/mass spectrometry. A, stereo view of the 2F_o − F_c electron density map at the active site of GspAF_H₂O₂. The electron density map, drawn at a contour level of 1σ, shows continuous density (in red) connected to the Sγ atom of Cys⁵⁹. This density was fitted with the oxygen atom of a sulfenic acid (-SOH) and the oxygen of a tightly hydrogen-bonded water molecule. Limited by the structure resolution, it is possible that the Asn¹⁴⁹ side chain can be flipped. B, schematic of the hydrogen bond network associated with the Cys⁵⁹ sulfenic acid in GspAF_H₂O₂. The sulfenic acid/water hydrogen bond is shown in red. Two other water molecules that contribute to the stability of the sulfenic acid are shown in blue. C, MALDI-MS-MS spectrum of the tryptic peptides that contained the Cys⁵⁹-dimedone adduct. Gsp amidase, after oxidation by H₂O₂, was treated with dimedone, digested with trypsin, and subjected to MALDI-MS. The dimedone-modified Cys⁵⁹-containing peptide (⁵⁷WQCVEFAR⁶⁴) has a molecular weight of 1176.6. That covalent dimedone modification of Cys⁵⁹ had occurred is evidenced by the y- and b-ions identified with asterisks.

FIGURE 5.

Conversion of (GspS)₂ to GSH and Spd by the Gsp amidase/GSH reductase couple. A, a MALDI-TOF spectrum that shows the molecular weights of the hydrolysis products after (GspS)₂ was treated with GspSA for 0 min (top), 10 min (middle), or 4 h (bottom). GSSG was the end product. B, the Gsp amidase/GSH reductase coupled assay. (GspS)₂ cannot be reduced by GSH reductase alone (■), but it is reduced when both GspSA and GSH reductase are present (▵). GSSG plus GSH reductase served as the positive control (●). GSH reductase alone served as the negative control (○). C, reaction scheme for the enzymatic conversion of (GspS)₂ to GSH and Spd by the Gsp amidase/GSH reductase couple.

FIGURE 6.

Conversion of GspSSPs to GSSPs by Gsp amidase-catalyzed hydrolysis. A, the (biotin-Gsp)₂ structure. B, schematic showing the preparation of biotinylated Gsp S-thiolated proteins (biotin-GspSSPs). Treatment of biotin-GspSSPs with GspSA generated GSH S-thiolated proteins. Treatment of biotin-GspSSPs with 2-ME generated unlabeled proteins. C, SDS-PAGE of biotin-GspSSPs. Right, Coomassie Brilliant Blue-stained proteins. Left, Western blot using an alkaline phosphatase-conjugated anti-biotin antibody. M_r, molecular weight markers. Lane 1, biotin-GspSSPs from an E. coli lysate. Lane 2, biotin-GspSSPs as in lane 1 that were treated with 20 mm 2-ME before SDS-PAGE. Lane 3, biotin-GspSSPs as in lane 1 that were treated with 5 μg of GspSA before SDS-PAGE.

FIGURE 7.

Glutaredoxin- and GspSA/glutaredoxin-null mutants exhibit hypersensitivities to exogenous H₂O₂. All strains were grown in M9 minimal medium until the cell densities reached 1.0 A₆₀₀. Then the cultures were treated with various concentrations of H₂O₂ for 1 h, and then 0.1 ml of each culture was transferred onto LB plates. The numbers of viable cells were then determined. Survival percentages (survivabilities) of ΔgrxA (▵, blue), ΔgrxB (▴, black), ΔgrxAΔgspSA (○, brown), and ΔgrxBΔgspSA (●, red) were calculated as ((CFU in the presence of H₂O₂)/(CFU in the absence of H₂O₂))/100. Survivability values (%) are the means of three replicates ± S.D. (error bars).

FIGURE 8.

The roles of Gsp and glutathionylspermidine synthetase/amidase in the intracellular redox regulation of E. coli. When exposed to reactive oxygen species (ROS), the active-site Cys⁵⁹ thiol of Gsp amidase is oxidized to sulfenic acid (1), which causes the inactivation of Gsp amidase (×) (2). Because Gsp synthetase is not affected by ROS, intracellular Gsp accumulates (3). Gsp may then scavenge harmful oxidants by forming Gsp-disulfides and other small molecule disulfide compounds (4A) and/or protecting protein thiols from oxidation by Gsp S-thiolation (4B). With the removal of the oxidative stresses, intracellular GSH and/or Gsp may rescue oxidized Gsp amidase, restoring amidase activity (4C). Reactivated Gsp amidase may hydrolyze Gsp to GSH and Spd or hydrolytically remove Spd from Gsp-disulfide or Gsp-modified proteins. Finally, the amount of Gsp returns to its basal level (5).