Redox regulation of 3'phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins

- C enzyme simultaneous of a mixed disulfide between glutathione and the active site Cys 239. method based on thiol-specific fluorescent alkylation and gel electrophoresis glutathionylated PAPS reductase reduced by glutaredoxins via monothiol-mechanism. glutathionylated gor - grx A grx B - grx C - cells inactive glutaredoxin better growing cells monothiol C12S or wild type Grx2. glutathionylation activity


Introduction
Sulfur is an ingredient of all living organisms. The first, most common form of sulfur in nature is inorganic sulfate, which needs to be further reduced in order to get incorporated in a living cell. Prototrophic bacteria for example use inorganic sulfate (SO 4 2-, S +VI ) as primary source for the biosynthesis of sulfur-containing amino acids and cofactors (1).
PAPS reductase (PR, EC 1.8.99.4) is composed of two identical subunits of 28 kDa. It is devoid of chromophores and contains a single cysteine per subunit in a highly conserved ECGLH-motif, identified as the redox-active center of the enzyme (2). Reduction of sulfate to sulfite by PR requires two electrons during which the cysteines of PR are oxidized to a disulfide. The oxidized enzyme is inactive and needs to be reduced for the reduction of PAPS to continue. Kinetic data (2)(3)(4) and the crystal structure of PR (5) implicate a ping-pong mechanism for its reduction. In the first step reduced PR catalyzes the reduction of PAPS, leading to oxidized PR, free sulfite and PAP (equation 1). Upon oxidation the enzyme dimer undergoes conformational changes leading to a significant decrease in apparent molecular weight and the inability to bind PAPS (2,3). In the second step, oxidized PR is reduced by thioredoxin (equation 2a) or glutaredoxin (equation 2b) with electrons from thioredoxin reductase (TR) and NADPH or from glutathione (GSH), glutathione reductase and NADPH. Thioredoxins and glutaredoxins are small (9 -14 kDa) ubiquitous proteins that utilize their two redox-active cysteines (CxxC-motif) to catalyze reduction of disulfides (6).
Whereas thioredoxins and glutaredoxins can reduce their substrates using both active site cysteines (dithiol mechanism) (7), glutaredoxins can also utilize the thiols from GSH in solution together with the glutaredoxin N-terminal cysteine (monothiol mechanism) (8). In addition to their ability of reducing intracellular disulfides, glutaredoxins may also reduce mixed disulfides forming between a protein thiol and oxidized GSH. This is a reaction not catalyzed by thioredoxins.
E. coli contains two thioredoxins (Trx1 and Trx2) and three glutaredoxins (Grx1, Grx2, and Grx3) (9). Trx1, Trx2, and Grx1 can reduce the disulfide that forms on ribonucleotide reductase 1a (RNR1a) upon the reduction of ribonucleotides with comparative efficiencies while Grx3 is only a weak reductant in vitro (10)(11)(12)(13). Trx1, Trx2 and Grx1 participate in the in vitro reduction of PAPS by PR (4). Other functions for Trx1 include the reduction of methionine sulfoxide via methionine sulfoxide reductase, while Trx2 participates in the antioxidant response as part of the OxyR regulon. Grx1 is also a member of the OxyR regulon but not Grx2 or Grx3 (14). With levels at least 10-fold higher than those of Grx1, Grx2 and Grx3 are highly abundant proteins in E. coli (15) and contribute up to 98 % of the GSH-dependent oxidoreductase activity using the disulfide between β-mercaptoethanol and GSH as substrate (HED assay) (11). Grx2 is an atypical glutaredoxin with a molecular mass of 23.4 kDa and structural similarities to mammalian GSH-S-transferases (16). Due to its high abundance (up to 1 % of total soluble protein) and catalytic efficiency it contributes to more than 80 % of the cellular GSH-mixed disulfides reducing activities (in the HED assay) (17,18). The enzyme is also highly active in the reduction of the mixed disulfide between glutathione and arsenate reductase (19). Grx2 is involved in the antioxidant response as mutants lacking Grx2 have increased levels of carbonylation of their intracellular proteins after exposure to hydrogen peroxide. Glutaredoxins and thioredoxins are not only direct antioxidants, but may also participate in the signal transduction of redox-induced cellular responses (overviews in: 20, 21).
Combined E. coli null mutants for glutathione reductase and the three glutaredoxins (gor -grxA -grxB -grxC -) barely grow on sulfate (S +VI ), but normally on sulfite (S +IV ) or methionine (S -II ) (22). As these mutants contain sufficient amounts of thioredoxin to reduce PR (15), this disturbed growth must represent some sort of inhibition of PR activity not based on the reduction of the enzyme's disulfide formed upon reduction of PAPS. Growth of gor -grxA -grxB -grxCcould be restored with monothiol or wild type Grx2 in trans, but not with the inactive C9S/C12S species (22). As Grx2 cannot reduce the disulfide of oxidized PR (4) this finding raises the possibility that the activity of PAPS reductase in vivo may be regulated by oxidized glutathione and glutaredoxins.

General methods
Materials, chemicals, and enzymes were purchased from different companies in the highest available purity. E. coli cells were transformed according to Hanahan (23). The concentration of proteins in crude extracts was determined as described by Bradford (24).

Growth of E. coli
To determine the redox-status of PR in cell free extracts and for immuno-

Western Blotting
The BioRad (Hercules) system was used according to the manufacturer's protocol.

Results
Growth properties of E. coli DHB4gor -grxA -grxB -grxC -Combined E. coli null-mutants for glutathione reductase and Grx1, Grx2, and Grx3 (DHB4gor -grxA -grxB -grxC -) barely grow in the presence of sulfate, but well in the presence of sulfite, cysteine, or methionine (22), (Fig. 1). Consistent with previous findings (22), transformants with the monothiol Grx2 grew faster and reached the highest optical density (OD 600 ) at stationary phase (2.4). The wildtype Grx2-containing cells reached an OD of 1.9, and the no-thiol Grx2-containing cells only an OD of 1.0 at stationary phase, as previously described for the non-transformed strain (22).

Redox-status of PAPS reductase in vivo
To investigate whether the limited growth of the null mutant was caused by an arrest of PR in the oxidized state, we determined the redox-state of the enzyme in vivo. PR is a homodimeric enzyme, whose active site is formed by an intermolecular dithiol-disulfide couple between the only cysteines at position 239 (2). As there are no other covalent links between the two subunits, the reduced and oxidized forms of the enzyme can be separated by non-reducing SDS-PAGE, where the reduced enzyme corresponds to an apparent Mr of 30 kDa and the oxidized to 60 kDa. No oxidized PR could be detected in the null-mutant transformed with the no-thiol Grx2 (Fig. 2, lanes 2 and 3), or in any other strain (data not shown). Therefore the inhibition of cell-growth in the particular strain was not caused by an arrest of PR in its oxidized conformation.

Reversible inhibition of PAPS reductase by oxidized glutathione
As the GSH/GSSG ratio in the gorstrain would be expected to shift more towards oxidized glutathione, we investigated whether GSSG affects PR activity. The enzyme was incubated with different amounts of GSSG before reduction of PAPS was performed with electrons delivered from Trx1, Trx reductase (TrxR) and NADPH (Fig. 3). Following the incubation with GSSG, the activity of PR decreased exponentially until no PAPS reduction was detectable. This inhibition pattern is characteristic for pseudo first order kinetics and suggested a covalent modification of PAPS reductase by GSSG as the basis for the inactivation, most likely due to the formation of a mixed disulfide between the enzyme and glutathione: The non-linear curve fitting of these results was made assuming pseudo-first order kinetics.
Calculated from the first-order rates obtained for the different GSSG-concentrations, the second-order rate constant was 80.4 ± 5.6 M -1 min -1 , indicating a rapid reaction between PAPS reductase and GSSG.
As the inhibition of PR was likely due to formation of a mixed disulfide between the active-site thiol and glutathione, we tried to restore enzymatic activity by addition of reductants. Addition of DTT or glutathione reductase could not restore the enzymatic activity of PR. When an additional glutaredoxin was added to the reaction mixture, the activity of the enzyme was restored to the former extent in less than 3 min (data not shown). All E. coli glutaredoxins (Grx1, Grx2, and Grx3) as well as their monothiol (CxxS)-mutants (Grx1C14S, Grx2C12S, Grx3C15S) were capable of reactivating PR in that time period.

Glutathionylation of cysteine 239
To confirm the glutathionylation of the active site cysteine 239, reduced and GSSGtreated PAPS reductase were analyzed by MALDI-TOF (Fig. 4). Tryptic digestion of the These results indicated that Cys 239 can form a mixed disulfide with glutathione.

Oxidation and reduction of glutathionylated PR
Highly purified oxidized and glutathionylated PR (1.93 ± 0.08 GSH per PR) were analyzed using thiol-specific alkylation with fluorescent 5-IAF and non-reducing SDS-PAGE (Fig. 5). Oxidized and glutathionylated PR exhibited virtually no fluorescence or activity (Fig. 5, lanes 1 and 2), whereas the Cys239-thiol of reduced PR was accessible for alkylation ( Fig. 5, lane 3) and active in single turn-over experiments (Fig. 5, panel I). The remaining portion of 30 kDa protein in the oxidized protein (lane 1) corresponds to redox-inactive Cterminal truncated protein without any cysteine (5). Reduced Grx1 reduced both the PRdisulfide and the mixed disulfide between PR and GSH in the presence and absence of GSH ( Fig. 5, lanes 4 and 5). Reduced monothiol Grx1C14S reduced the two PR species in the presence of GSH, but formed a stable mixed disulfide with PR in the absence of GSH (Fig. 5,   lanes 6 and 7). Grx3, as well as its monothiol mutant Grx3C15S, reduced the glutathionylated form of PR in the presence of GSH (Fig. 5 A, lane 9 and 11), but not the intramolecular PR disulfide (Fig. 5 A, lane 8 and 10). As seen in the reactivation assays, DTT or GSH alone could not reduce glutathionylated PR, as they did not increase the amount of free thiols of the glutathionylated form (Fig. 5 lanes 2 and 12). Remarkably, in all preparations the existence of free thiols in PR corresponded to protein in the active conformation (panels I and II).

Glutathionylation of PR in vivo
Was glutathionylated PR the reason for the limited growth of the gor -grxA -grxB -grxCstrain in M9 media free of reduced sulfur? Antibodies raised against GSH-moieties on BSA (31) reacted specifically with glutathionylated PR and showed no cross reactivity with reduced or oxidized PR (Fig. 6 B, lanes 1 -3). No glutathionylated PR was detected in extracts from gor -grxA -grxB -grxCtransformed with Grx2C12S (Fig. 6, lane 6). The mixed disulfide species was detected in extracts from transformants encoding wild type Grx2 or the no thiol Grx2C9S/C12S mutant (Fig. 6, lanes 4 and 5). From the density of the bands on the blot, 20 % of the PR in the pISCGrx2 transformants was glutathionylated (2.8 ng out of 14 ng detected). 40 % was the estimation for the pISCGrx2C9S/C12S-transformants (6.2 ng out of 15.9 ng detected). In view of the findings of this work, a more complete picture emerges for the catalytic mechanism of PR (Fig. 7). In a 'normal'-reducing cell environment reduced PR is a homodimer. PAPS can bind to this reduced form (Fig. 7, R), to yield sulfite, PAP and oxidized PR with an intra-molecular disulfide bridge between the active site cysteines (Cys 239) ( fig. 7, reaction 1). Dimeric oxidized PR migrates on SDS PAGE with an apparent molecular weight (60 kDa) higher than that of the reduced form (30 kDa). The disulfide of the oxidized enzyme can be reduced by Trx1, Trx2 or Grx1 but not the other glutaredoxins ( Fig. 7, reaction 2) (4,44). If the intracellular environment is somewhat oxidizing (e.g. the gorstrain), a mixed disulfide may form between Cys 239 and glutathione rendering the enzyme inactive. All glutaredoxins can catalyze the reduction of this mixed disulfide (Fig. 7,   reaction 4).

Discussion
Formation of protein-glutathione mixed disulfides is of physiological relevance for E.
coli. Up to 2 % of the total glutathione content (10 -20 µM) is in the form of protein-mixed disulfides and this value can be increased, as for example in trxA -grxAmutants (5 -7 %) (45). In mammalian cells, extensive glutathionylation of protein substrates has been identified to include chaperons, cytoskeletal proteins, cell cycle regulators and enzymes participating in the intermediary metabolism (46). Such a study has not been performed yet for E. coli.
Glutaredoxins and their monothiol activity would be the expected molecules to key regulate deglutathionylation reactions and reverse related changes in biological activity. The identification and characterization of further proteins that undergo reversible Sglutathionylation and are specifically related to the glutaredoxin species will be necessary for a deeper understanding of cellular redox regulation and signaling.   E. coli DHB4gor -grxA -grxB -grxCwas transformed with plasmids expressing wildtype Grx2
Cultures of 500 ml M9-medium supplemented with Leu, Ile (33 mg l -1 ), and 100 µg ml -1 ampicillin were inoculated to an optical density (A 600 ) of 0.14 using overnight cultures grown in LB-medium that were previously washed twice with M9-medium. The expression of Grx2 and the C12S and C9S/C12S mutants was induced by 0.1 % arabinose.  Incubation time versus lg(activity). The non-linear curve fitting was done assuming pseudo first order kinetics.

Figure 4 -MALDI-TOF analysis of tryptic fragments from reduced (A) and glutathionylated PAPS reductase (B)
The reduced and glutathionylated peptide masses corresponding to the cys 239-containing peptides are marked with bold/italic characters.