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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 8404–8409, July 1998 Plant Biology

Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5؅-adenylylsulfate

JULIE-ANN BICK*, FREDRIK ÅSLUND†,YICHANG CHEN*, AND THOMAS LEUSTEK*‡

*Biotech Center and Plant Science Department, Rutgers University, New Brunswick, NJ 08901-8250; and †Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115

Edited by Bob B. Buchanan, University of California, Berkeley, CA, and approved May 14, 1998 (received for review November 21, 1997)

ABSTRACT 5؅-Adenylylsulfate (APS) reductase (EC the basis of their similar catalytic requirements and molecular 1.8.99.-) catalyzes the reduction of activated sulfate to sulfite weights (4, 6). The major difference is that the product of the in plants. The evidence presented here shows that a domain of sulfotransferase is believed to be an organic thiosulfate (1), the is a (GSH)-dependent reductase that whereas the reductase may produce sulfite (4), although the functions similarly to the . The actual reaction product has not been determined for either APR1 cDNA encoding APS reductase from Arabidopsis thali- enzyme. APS reductase is distinguished from the cysH product ana is able to complement the cysteine auxotrophy of an by its preference for APS over PAPS and by its ability to Escherichia coli cysH [3؅-phosphoadenosine-5؅-phosphosul- function in an E. coli thioredoxin͞glutaredoxin double mutant fate (PAPS) reductase] mutant, only if the E. coli strain (4). These properties may be due to its unusual two-domain produces glutathione. The purified recombinant enzyme structure, consisting of a reductase domain (R domain) at the (APR1p) can use GSH efficiently as a hydrogen donor in vitro, amino terminus, showing homology with the cysH product; and Ϸ showing a Km[GSH] of 0.6 mM. Gene dissection was used to a carboxyl-terminal region (C domain), showing homology express separately the regions of APR1p from amino acids with and glutaredoxin. This structure suggests that 73–327 (the R domain), homologous with microbial PAPS a dithiol– transhydrogenase mechanism may be in- reductase, and from amino acids 328–465 (the C domain), volved in APS reduction. It should be noted that the APR- homologous with thioredoxin. The R and C domains alone are encoded enzyme shows no sequence homology with the APS inactive in APS reduction, but the activity is partially restored reductase from dissimilatory sulfate-reducing bacteria, which by mixing the two domains. The C domain shows a number of is an iron–sulfur flavoenzyme (7). To avoid confusion we activities that are typical of E. coli glutaredoxin rather than propose that the enzyme described in this report be referred thioredoxin. Both the C domain and APR1p are highly active to as the plant-type APS reductase. in GSH-dependent reduction of hydroxyethyldisulfide, cys- belonging to the thioredoxin superfamily contain a tine, and dehydroascorbate, showing a Km[GSH] in these assays reactive Cys pair that undergo reversible disulfide bond for- Ϸ of 1 mM. The R domain does not show these activities. The mation as electrons are transferred to a variety of C domain is active in GSH-dependent reduction of insulin (3). Thioredoxin is specifically reduced by NADPH or ferre- and , whereas APR1p and doxin-dependent (8). Glutaredoxin, a R domain are inactive. The C domain can substitute for related cofactor, is specifically reduced by glutathione (GSH), glutaredoxin in vivo as demonstrated by complementation of which is itself maintained in a reduced state by NADPH- an E. coli mutant, underscoring the functional similarity dependent (9). The specificity for GSH between the two . lies in a glutathione in glutaredoxin that is absent from thioredoxin (10). Plants and microorganisms are able to reduce sulfate to sulfide In this report, evidence is presented that GSH may be the for synthesis of the thiol group of cysteine. Sulfate is first electron source used by APS reductase. This result is signifi- activated by ATP sulfurylase, forming 5Ј-adenylylsulfate cant because GSH was proposed to be the natural substrate of (APS). APS can be phosphorylated by APS kinase, forming APS sulfotransferase in plants (11). Further, enzyme dissec- 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS). Depending tion revealed that the C domain of APS reductase shows upon the organism, either APS or PAPS can be used for sulfate GSH-dependent transhydrogenase activities that are charac- reduction. In general, it is thought that and fungi teristic of glutaredoxin. Finally, we report that the R and C use PAPS, whereas photosynthetic eukaryotes use APS (1). domains, expressed as separate proteins, are not active in APS However, the APS pathway is not universally accepted, and it reduction, but the activity is reconstituted when they are mixed has been argued that plants may reduce sulfate as do micro- together. This result establishes the independent but interac- organisms by means of PAPS reductase (2). This enzyme, tive catalytic roles played by the APS reductase domains. encoded by cysH in Escherichia coli, is dependent on the cofactors thioredoxin or glutaredoxin (3). Key evidence supporting an APS-dependent pathway was MATERIALS AND METHODS obtained by cloning of three individual cDNAs (APR1, -2, and Reagents and General Methods. Nuclease P1 (catalog. no. -3; accession nos. U43412, U56921, and U56922, respectively) N8630), Spirulina thioredoxin (T3658), insulin (I5500), and for APS reductase (EC 1.8.99.-) from (4, glutathione reductase (G4759) were from Sigma. Bovine thi- 5). This enzyme was termed a ‘‘reductase’’ because of its homology with the encoded by cysH; however, it is This paper was submitted directly (Track II) to the Proceedings office. likely identical to the enzyme termed APS sulfotransferase, on Abbreviations: GSH, reduced glutathione (when the reduced form is not specifically being referred to, the term ‘‘glutathione’’ is used); ␥ ␥ Ј Ј The publication costs of this article were defrayed in part by page charge -EC, -glutamylcysteine; APS, 5 -adenylylsulfate; PAPS, 3 - phosphoadenosine-5Ј-phosphosulfate; DHA, dehydroascorbate; payment. This article must therefore be hereby marked ‘‘advertisement’’ in HED, hydroxyethyldisulfide. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: Biotech Center, 59 © 1998 by The National Academy of Sciences 0027-8424͞98͞958404-6$2.00͞0 Dudley Road, Rutgers University, New Brunswick, NJ 08901-8250. PNAS is available online at http:͞͞www.pnas.org. e-mail: [email protected].

8404 Downloaded by guest on October 1, 2021 Plant Biology: Bick et al. Proc. Natl. Acad. Sci. USA 95 (1998) 8405

oredoxin reductase was purchased from IMCO (Stockholm). Table 1. E. coli strains E. coli thioredoxin and thioredoxin reductase were the kind Strain Relevant genotype Origin gift of Charles Williams (Veterans Affairs Medical Center, Ann Arbor, MI). E. coli ribonucleotide reductase was the kind JM96 cysH56 CGSC* gift of Joanne Stubbe (Massachusetts Institute of Technology, KL39 P1kcϩ CGSC* Cambridge). [35S]PAPS was purchased from New England JF518 gshB::kan (19) Nuclear. [35S]APS was prepared by dephosphorylating JTG10 gshA::Tn-miniKan (20) [35S]PAPS (4). A304 trxB15::kan (21) Bacteriological media were prepared as described by Miller TL1 cysH56, gshB::km This study (12). Sambrook et al. (13) was followed for nucleic acid TL2 cysH56, gshA::km This study methods and Laemmli (14), for denaturing protein gel elec- TL3 cysH56, trxB15::km This study trophoresis. Concentrations of pure proteins were measured DHB4 ⌬(ara–leu)7697 (22) from the difference in absorbance at 280 and 260 nm (15), and FA87 ⌬trxA, trxC, grxA::km † the concentrations of proteins in crude extracts were measured pBAD39-trxC by the Bradford method (16). ⌬(ara–leu)7697 Construction of Expression Plasmids and Purification of FA47 ⌬trxA, grxA::km † Recombinant Proteins. For protein purification the Novagen ⌬(ara714–leu)::Tn10 S-Tag system was used. The APR1-encoded protein (APR1p) *Coli Genetic Stock Center (Yale University). expression plasmid (pET-APR1) was prepared by cloning a †E. J. Stewart, F.Å, and J. Beckwith, unpublished work. 1420-bp EcoRI–SalI fragment from the APR1 cDNA into pET-30a. The C-domain expression plasmid (pET-C) was with pBAD-C or pBAD33-grxA as a positive control. These prepared by cloning the 380-bp EcoRV–SalI fragment of APR1 strains are derived from E. coli strain DHB4. FA87 carries a into pET-30a. A plasmid was prepared from which C-domain complementing TrxC plasmid containing the counterselect- expression is regulated by an arabinose-inducible promoter able wild-type rpsL allele. After transformation with either (pBAD-C) by cloning the Ϸ400-bp XbaI–HindIII fragment pBAD-C or pBAD33-grxA the transformants were counters- from pET-C into pBAD33 (17). The R-domain expression elected on medium containing streptomycin sulfate (selection plasmid (pET-R) was prepared by removing the 380-bp for colonies that have lost the TrxC plasmid). The colonies EcoRI–SalI C-domain fragment from pET-APR1. pET-R were then tested for arabinose-dependent complementation carries a 1040-bp R-domain fragment and produces a protein on medium containing 0.2% (wt͞vol) arabinose. Rich medium that includes up to amino acid 348 in APR1p. The recombinant was used for FA87 and minimal medium lacking cysteine was C domain includes amino acids 349 to 465 of APR1p. The used for FA47. Arabinose-dependent expression of the C APR2 expression plasmid was prepared by cloning a 1450-bp domain was confirmed by immunoblotting with an S-Tag EcoRI fragment from APR2 into pET-30a. The APR3 expres- antibody. sion plasmid was prepared by cloning a 1500-bp EcoRI frag- Enzyme Assays. Unless indicated otherwise, the unit of ment from APR3 into pET-30b. enzyme activity is defined as ␮mol of product formed per min. BL21(DE3)plysS was transformed with the pET expression Apparent Km values were calculated by least-squares nonlinear plasmids. Transformants were grown at 37°C to an optical regression analysis (24). All values were determined from at density (at 600 nm) of 0.6–0.8 and expression was induced with least three independent experiments. 1 mM isopropyl ␤-D-thiogalactoside (IPTG) for 3 h. The cells APS reductase was measured at 30°C (4). The 100-␮l reaction ⅐ from 100-ml culture were resuspended in 10 ml of 150 mM mixture contained 100 mM Tris HCl (pH 8.5), 500 mM Na2SO4, NaCl͞0.1% (vol͞vol) Triton X-100͞20 mM Tris⅐HCl, pH 7.5, 1 mM EDTA, 25 ␮M[35S]APS (Ϸ500 Bq⅐nmolϪ1), pure APS and were then lysed by a freeze–thaw cycle followed by reductase or protein extract, and a hydrogen donor (DTT, GSH, sonication. The lysate was centrifuged (14,000 ϫ g for 10 min) cysteine, ␥-EC, or 1 mM NADPH and 6 units of thioredoxin and the supernatant was filtered through a 0.4-␮m-pore mem- reductase). The reaction was started by addition of the hydrogen brane. The lysate was incubated with 2 ml of S-protein agarose donor. The amount of sulfur dioxide formed in a reaction lacking for 30 min. The agarose was washed with 5 vol of lysis buffer the hydrogen donor (the background rate) was subtracted from and the bound protein was released by overnight digestion with the complete reaction mix to obtain the catalytic rate. To avoid enterokinase, which was subsequently removed by using large changes in substrate concentration the incubation time and Ekapture (Novagen) agarose. The supernatant containing the protein concentration were adjusted so that the amount of purified recombinant protein was dialyzed against 100 mM product formed was at least 5-fold above the background but no Tris⅐HCl, pH 8.5, and stored at Ϫ70°C. On average Ϸ800 ␮g more than 10% of the substrate in the reaction. In some cases the of recombinant protein was recovered. reaction was allowed to progress to completion to confirm that Glutaredoxin was purified from E. coli DHB4 transformed the enzyme can convert all the substrate to product. with pBAD-grxA (grxA cloned into pBAD18; ref. 17). The GSH-dependent reduction of hydroxyethyldisulfide (HED) purification was carried out as in ref. 18, except that ultrafil- or cystine was measured in a 500-␮l reaction mixture contain- tration replaced the gel filtration step. No contaminating ing 0.8 mM HED or cystine, 0.35 mM NADPH, 2 units of proteins were visible on an overloaded SDS͞PAGE gel. glutathione reductase, 1.5 mM EDTA, 100 mM Tris⅐HCl (pH Strain Construction and Complementation Assays. The E. 8.0), and GSH (25). APRp was added, and the absorbance at coli strains used in this study are described in Table 1. TL1, -2, 340 nm was measured continuously for 2 min at 24°C. and -3 were prepared by transduction (12) of JM96 with phage GSH-dependent reduction of dehydroascorbate (DHA) was P1 lysates prepared in JF518, JTG10, and A304, respectively. measured in a 500-␮l reaction mixture containing 2.0 mM Transduction was carried out with infective P1kc prepared by DHA, 0.2 mM NADPH, 2 units of glutathione reductase, 0.27 growing KL39 at 42°C. M9 medium with leucine, tryptophan, mM EDTA, 50 mM sodium phosphate (pH 6.9), and GSH histidine, arginine, and thiamin, with or without cysteine, was (26). The mixture, lacking DHA and APRp, was preincubated used for complementation analysis of cysH strains with for 5 min. DHA and then APRp were added, and the p␭YES-APR1 (4). The inocula for the experiment in Fig. 1 absorbance at 340 nm was measured continuously for 2 min at were transferred once on cysteine-free medium. Glutathione, 24°C. ␥-glutamylcysteine (␥-EC), and cysteine were measured by In the HED, cystine, and DHA assays the rate of NADPH HPLC using fluorescent labeling with monobromobimane oxidation in the absence of APRp served as the background (23). Complementation of FA47 and FA87 was carried out rate. All the reductase assays were tested with 6 units of Downloaded by guest on October 1, 2021 8406 Plant Biology: Bick et al. Proc. Natl. Acad. Sci. USA 95 (1998)

thioredoxin reductase replacing GSH and glutathione reduc- tase, but no activity was observed with this alternate reducing system. The reduction of insulin disulfides (27) was measured in a 600-␮l reaction mixture containing 2 mM EDTA, 100 mM sodium phosphate (pH 6.5), 0.13 mM insulin, 20 ␮M thiore- doxin, GrxA or APRp, and a hydrogen source (1 mM DTT; 1 mM NADPH and 6 units of thioredoxin reductase; or 5 mM GSH, 1 mM NADPH, and 6 units of glutathione reductase). The increase in absorbance at 650 nm was monitored contin- uously at 24°C. The reduction of ribonucleotide reductase disulfides was determined by measuring its activity (28). The 400-␮l reaction mixture contained 30 mM Hepes (pH 7.6), 10 mM MgCl2, 1.5 mM EDTA, 0.5 mg⅐mlϪ1 BSA, 1.3 mM ATP, 0.05 unit of ribonucleotide reductase, 1 mM NADPH, cofactor (thiore- doxin, GrxA, or APRp), and a reducing system (6 units of thioredoxin reductase or 5 mM GSH and 6 units of glutathione reductase). Ribonucleotide reductase was prereduced by in- cubation for 60 min on ice with 5 mM DTT. The DTT was FIG. 1. Complementation of E. coli cysH by plant APS reductase removed by gel filtration on Sephadex G-25. The reaction was requires glutathione. WT refers to the cysH strain carrying wild-type started by addition of 1 mM CDP. The decrease in absorbance alleles for gshA, gshB, and trxB. The others are cysH strains carrying the at 340 nm was monitored continuously at 24°C. designated mutation. The relevant medium nutrient composition is indicated above and below the photographs. The cultures were incu- bated for 48 h at 30°C. Thiol compounds and APS reductase activity RESULTS were measured in extracts prepared from the cells grown on ϩcysteine medium. The concentrations of thiol compounds are given below in Determination of the Hydrogen Donor for APS Reductase. nmol per g of fresh weight and APS reductase activity is given in The APR cDNAs encoding APS reductase are able to com- pmol⅐minϪ1 per mg of protein. WT: GSH ϭ 383, ␥-EC not detected, plement the cysteine auxotrophy of an E. coli cysH mutant cysteine not detected, and APS reductase ϭ 215. trxB: GSH ϭ 575, strain (4). The structure of APS reductase raised the question ␥-EC not detected, cysteine not detected, and APS reductase ϭ 4950. gshA: GSH not detected, ␥-EC not detected, cysteine ϭ 10, and APS of what reducing system is used during complementation of ϭ ϭ ␥ ϭ ϭ cysH. The availability of glutathione and thioredoxin reductase reductase 210. gshB: GSH 6, -EC 70, cysteine 13, and APS reductase ϭ 225 mutants of E. coli allowed us to explore this question. Mutant ␥ alleles for gshA ( -glutamylcysteine synthetase), gshB (gluta- Analysis of the Function of APS Reductase Domains. The thione synthetase), and trxB (thioredoxin reductase) were function of the APS reductase domains was studied by ex- separately introduced into the cysH strain, and the derivatives pressing and purifying each as a separate recombinant were used to test the ability of APR1 to complement cysH. Fig. polypeptide. The preparations used in the experiments are 1 shows that even though there is APS reductase activity in all shown in Fig. 2, lanes 3 and 5. The isolated R (reductase) and the strains APR1 is able to complement cysH only if the strain C (carboxyl-terminal) domains were found to have low APS contains glutathione. Pure recombinant APS reductases (APR1p, APR2p, and reductase activity, but this activity was not catalytic, because it APR3p) were used to study the hydrogen donor requirement was not proportional to the amount of protein added to the in vitro. The APR1p preparation is shown in Fig. 2, lane 2. reaction (Fig. 4). DTT alone or in combination with thiore- APR2p and APR3p showed a similar size and level of purity doxin or glutaredoxin did not promote APS reductase activity (not shown). Fig. 3 shows the APS reductase activity of APR1p for either domain. When the R and C domains were combined, with three different electron donors. GSH is as effective as APS reductase activity was reconstituted, and this activity DTT in promoting APS reduction, whereas 2-mercaptoetha- nol is ineffective. At high concentrations GSH is slightly inhibitory. The GSH titration shows that Vmax of APR1p is 0.18 ␮mol⅐minϪ1⅐mgϪ1 achieved with 8–10 mM GSH. Similar re- sults were obtained with the other APS reductases, although APR2p shows a significantly greater Vmax (Table 2). Irrespec- tive of their specific activity, all the APR proteins show a similar Km value for GSH ranging from 0.6 to 1.2 mM. Cysteine and ␥-EC were also tested as hydrogen sources for the APR proteins (data not shown). Cysteine did not function as a ␥ hydrogen donor, but the Vmax achieved with -EC was com- parable to that with GSH, although the Km[␥-EC] was 9-fold greater than Km[GSH]. NADPH in combination with E. coli or bovine thioredoxin reductase and with or without E. coli or FIG. 2. Purified recombinant APR1p, R domain, and C domain. Spirulina thioredoxin was unable to promote APS reductase Lane 1, molecular mass standards; lane 2, 2 ␮g of APR1p; lane 3, 2 activity in any of the APR enzymes. A variety of conditions ␮g of R domain; lane 4, molecular mass standards; and lane 5, 1 ␮g were tested. In particular, it was found that under the APS of C domain. Lanes 1–3 and 4–5 were run on 10% and 12.5% polyacrylamide gels, respectively. The mass in kDa of the recombinant reductase assay conditions (pH 8.5, 500 mM Na2SO4) the ͞ ͞ proteins predicted from the amino acid sequence and based on gel NADPH thioredoxin thioredoxin reductase system retains ϭ Ϸ mobility (in parentheses) is as follows: APR1p 53.7 (51 and 49); R 80% of its activity as measured by using the insulin disulfide domain ϭ 40.1 (41 and 39.5); and C domain ϭ 13.7 (12.8). The reduction assay, so APS reduction would have been observed migration of APR1p and R domain as a doublet is likely due to if the APR enzymes were able to use this system as a hydrogen inefficient cleavage with enterokinase. The molecular masses of source. standards in kDa are 200, 97.4, 68, 43, 29, 18.4, and 14.3. Downloaded by guest on October 1, 2021 Plant Biology: Bick et al. Proc. Natl. Acad. Sci. USA 95 (1998) 8407

FIG. 3. Hydrogen donor requirement of APS reductase. The ᮀ ␮ FIG. 4. APS reductase activity of isolated APR1p domains. ,R activity from 0.60 g of APR1p was measured with various amounts domain; ■, C domain. Various amounts of C domain were added to a E F ᮀ of DTT ( ), GSH ( ) or 2-mercaptoethanol ( ). The assays were reaction with 0.5 ␮l(E)or1␮l(F) of R domain. The reaction mixtures incubated for 20 min. Each point is the mean of three assays. Similar contained 10 mM GSH. The concentration of the protein solutions was results were obtained with four different APR1p preparations. 20 pmol⅐␮lϪ1.

showed the features that are expected of a reaction catalyzed as HED, cystine, and DHA (24, 25). The C domain was found by two enzymes (Fig. 4). At a fixed level of R domain the to be very active in HED, cystine, and DHA reduction (Table reaction rate is directly proportional to the amount of C 2), with activities that are comparable to the activity of domain in the reaction up to a point beyond which the rate did glutaredoxin. APR1p is also active in these reactions, but the not increase further. At the highest levels of C domain the R R domain is inactive. When GSH and glutathione reductase domain is limiting, the reaction rate being proportional to the are replaced with thioredoxin reductase the C domain is amount of R domain in the reaction. A specific activity for the Ϫ Ϫ unable to catalyze the reduction of the small molecules. The R domain of approximately 0.022–0.025 ␮mol⅐min 1⅐mg 1 can Ϸ ͞ ͞ Km[GSH] of the C domain and APR1p in these assays is 1 mM, be calculated from the data. This is 1 6to1 8 the Vmax of the similar to the Km[GSH] of the APR enzymes in the APS intact APR1p enzyme. The molar ratio of R and C domains reductase assay. In total, the results suggest that it is the C needed to achieve maximal activity is approximately 1:60 domain of APS reductase that mediates the interaction of the compared with a 1:1 ratio for the intact enzyme. It is tempting enzyme with GSH by functioning as a glutaredoxin. to speculate that the high molar ratio and decreased catalytic Both thioredoxin and glutaredoxin have the ability to reduce efficiency is because of inefficient interaction of the separated the disulfides of insulin (27). In the presence of DTT as a R and C domains, but it is also possible that expression as hydrogen source the C domain was found to be able to reduce recombinant protein fragments has resulted in preparations insulin, whereas the R domain and APR1p were found to be that are only partially active. However, the results show that the inactive (Fig. 5). A similar level of activity for the C domain APS reductase domains play independent and interactive roles and GrxA could be obtained when DTT was replaced with in catalysis. GSH, NADPH, and glutathione reductase, whereas thiore- Glutaredoxin Function of the C Domain of APS Reductase. doxin was inactive with this reducing system (not shown). The homology of the C domain with disulfide-active cofactors, Another activity associated with thioredoxin and glutaredoxin coupled with the finding that APS reductase uses GSH as a is the ability to serve as a hydrogen donor for ribonucleotide source of protons, suggests that the C domain may function as reductase (27). The C domain, in combination with GSH, glutaredoxin, a GSH-disulfide (3, 9). Glutare- doxin has several characteristic activities, including the ability to catalyze GSH-dependent reduction of small molecules such

Table 2. Reductase assays and kinetic constants

Km[GSH], Vmax, Assay Protein mM ␮mol⅐minϪ1⅐mgϪ1 APS APR1p 0.6 0.18 APR2p 1.2* 3.10 APR3p 0.9* 0.21 HED APR1p 1.2 21.2 C 1.2 60.5 RNANA Cystine APR1p 1.2 41.1 C 1.1* 54.3 RNANA DHA APR1p 1.1* 52.8 C 1.2* 62.4 FIG. 5. The C domain can reduce the disulfides of insulin. The activity was measured with 20 ␮M of the enzyme indicated on the RNANA graph. The thioredoxin is from E. coli. The activity of the R domain Assay refers to the substrate reduced; Km[GSH] was measured at and APR1p was not greater than the spontaneous rate in a reaction saturating level of the second substrate indicated in the assay column. lacking protein. The assay measures the precipitation of the insulin ␤ Ϫ1 Vmax is in ␮mol⅐min per mg of protein; NA, no activity or unable to chain, detected as an increase in turbidity at 650 nm. A reaction with be calculated. All values were calculated from four independent data 20 ␮M glutaredoxin (Grx1) gave a curve that lies between the curves .(indicates SD 10–20% of value; all others less than 10% of value. of thioredoxin and C domain (not shown ء .sets Downloaded by guest on October 1, 2021 8408 Plant Biology: Bick et al. Proc. Natl. Acad. Sci. USA 95 (1998)

NADPH, and glutathione reductase, is able to serve as a Table 3. Ribonucleotide reductase activity with C domain, hydrogen donor for ribonucleotide reductase (Fig. 6). The glutaredoxin, or thioredoxin reaction is dependent on the presence of CDP, the ribonucle- Reducing Activity, otide reductase substrate, and the rate is proportional to the Protein system nmol͞10 min amount of C-domain added. This result indicates that under ͞ these conditions (up to 0.8 mM C domain and excess ribonu- C domain GSH GR 24.4 ͞ cleotide reductase) the activity of the C domain limits the GrxA GSH GR 172.4 ͞ reaction rate. Table 3 shows that with GSH, glutathione Thioredoxin GSH GR NA reductase, and NADPH as the reducing system the C domain C-domain TR NA and glutaredoxin are able to promote ribonucleotide reductase GrxA TR NA activity but thioredoxin is unable to do so. The specific activity Thioredoxin TR 93.8 of the C domain is Ϸ1͞7 that of GrxA. With thioredoxin C domain, GrxA, and E. coli thioredoxin were 0.8 ␮M. GSH͞GR reductase and NADPH as the hydrogen source only thiore- refers to glutathione reductase and GSH, and TR refers to the ϩ doxin is able to promote ribonucleotide reductase activity. thioredoxin reductase system. Activity is in nmol of NADP per 10 Taken together, these results demonstrate that the C domain min. NA, no activity. is able to catalyze GSH-dependent reduction of protein dis- ͞ ulfides in a fashion similar to that of glutaredoxin. domain and GrxA with respect to specificity and or reducing As with the insulin reduction assay, APR1p and the R properties. domain are inactive with ribonucleotide reductase. This result is in contrast to the results with small molecules, where APR1p DISCUSSION showed activity comparable to that of the isolated C domain. Thus it appears that reduction of protein disulfides by the C Evidence is presented that APS reductase uses GSH as a domain is blocked when it is physically associated with the R hydrogen donor and that its C domain shows properties that domain. resemble those of glutaredoxin. The evidence also shows that The ability of the C-domain to serve as a protein disulfide the catalytic mechanism of APS reductase depends on the reductase was tested by a complementation assay using an E. interaction of two domains with distinct functions. coli strain carrying mutations in trxA, trxC, and grxA. Strain The simplest explanation for the ability of APR1 to com- FA87 is absolutely dependent on a complementing plasmid plement the cysH mutation only if the strain produces gluta- encoding a functional disulfide reductase (E. J. Stewart, F.Å., thione is that APS reductase uses GSH as a hydrogen donor. and J. Beckwith, unpublished work). For example, it can grow The key finding that bolsters this hypothesis is that the C if it carries a GrxA-expression plasmid but is unable to grow domain of APS reductase functions like glutaredoxin, a well with GrxC, another E. coli glutaredoxin, which is a poor characterized GSH-dependent oxidoreductase. disulfide reductant compared with GrxA (29). Thus, FA87 Glutaredoxin serves as a cofactor for a number of reduc- offers a convenient way of assessing the in vivo disulfide tases, including ribonucleotide reductase (3); thus, knowledge reducing capacity of a test protein. The C-domain plasmid was of its function could be invaluable in proposing a catalytic able to complement strain FA87 in an arabinose-dependent mechanism for APS reductase. There are two proposed mech- manner, demonstrating in vivo the disulfide reductase activity anisms for glutaredoxin. The first, for reduction of protein of the C domain. However, the growth rate was slower than disulfides, requires the two Cys residues, and an with the GrxA plasmid. This difference could reflect the lower intramolecular disulfide with the substrate protein is formed as efficiency of the C domain to serve as a reductant of ribonu- an intermediate (18). In the case of ribonucleotide reductase, cleotide reductase (Table 3). We also tested whether the glutaredoxin reduces a disulfide, which charges the reductase C-domain plasmid can complement another in vivo redox with the protons needed for ribonucleotide reduction (30). In function attributed to thioredoxin and glutaredoxin, the ability this model, it is possible that the C domain may function in to serve as a hydrogen donor for PAPS reductase (strain transferring protons to the R domain, which then directly FA47). The C-domain plasmid was unable to complement this catalyzes APS reduction. The R domain of all the APR strain, indicating that it is unable to drive PAPS reductase proteins contain two Cys pairs that could be reduced by the C activity. This result may reflect a difference between the C domain (CEPC, amino acids 294–297; and CC, amino acids 202–203 in APR1p). However, the mechanism is unlikely to be a simple transfer of protons, because E. coli thioredoxin or glutaredoxin was unable to substitute for the C domain in promoting APS reduction by the R domain. This finding suggests that there is specificity in the interaction between the R and C domains. It is intriguing that APR1p is unable to catalyze insulin precipitation or serve as a hydrogen donor for ribonucleotide reductase, whereas it is able to reduce small disulfide mole- cules. This could be due to steric inhibition of the C domain by the R domain, or it might reflect a fundamental difference in catalytic mechanism between protein disulfide reduction and the reduction of small molecules. Indeed, reduction of small molecule disulfides by glutaredoxin is thought to proceed by a mechanism different from reduction of protein disulfides. This mechanism requires only one of the two active site Cys residues, the reactive Cys (Cys-14 in T4 Grx) and involves the formation of an intermediate mixed disulfide with glutathione FIG. 6. C domain can serve as the hydrogen donor for ribonucle- otide reductase. The rate of NADPH oxidation is presented as the (18). The high activity of APS reductase in reducing small change in absorbance at 340 nm. The assay was carried out with three molecules raises the interesting possibility that the C domain different concentrations of C domain, indicated on the graph in ␮M. could function in reducing S-sulfoglutathione generated by the The background rate was a reaction with 0.8 ␮M C domain but lacking R domain or S-sulfo derivatives of the R domain. Another the ribonucleotide reductase substrate CDP. intriguing possibility is that APS reductase could play a role Downloaded by guest on October 1, 2021 Plant Biology: Bick et al. Proc. Natl. Acad. Sci. USA 95 (1998) 8409

outside sulfate assimilation, in reducing a variety of small 2. Schiffmann, S. & Schwenn, J. D. (1994) FEBS Lett. 355, 229–232. disulfide molecules or S-sulfo compounds generated sponta- 3. Holmgren, A. (1989) J. Biol. Chem. 264, 13963–13966. neously when sulfite, a toxic and reactive compound, accumu- 4. Setya, A., Murillo, M. & Leustek, T. (1996) Proc. Natl. Acad. Sci. lates in plastids, such as during sulfur dioxide exposure (31). USA 93, 13383–13388. Thioredoxin and glutaredoxin share similar active sites and 5. Gutierrez-Marcos, J., Roberts, M. A., Campbell, E. I. & Wray, have similar secondary structures (3). However, it is the GSH J. L. (1996) Proc. Natl. Acad. Sci. USA 93, 13377–13382. binding site of that is a key structural feature of 6. Kanno, N., Nagahisa, E., Sato, M. & Sato, Y. (1996) Planta 198, this class of cofactors (10, 32). Alignment of the C-domain 440–446. 7. Speich, N., Dahl, C., Heisig, P., Klein, A., Lottspeich, F., Stetter, sequence with the glutaredoxins reported in ref. 10 indicates K. O. & Tru¨per, H. G. (1994) Microbiology (Reading, U.K.) 140, that it contains many of the amino acids that constitute the 1273–1284. conserved GSH binding site. Moreover, the predicted second- 8. Buchanan, B. B., Schu¨rmann, P., Decottignies, P. & Lozano, ary structure of the C domain closely matches that of the R. M. (1994) Arch. Biochem. Biophys. 314, 257–260. glutaredoxins. This match suggests that the C domain may 9. Fuchs, J. A. (1989) in Glutathione—Chemical, Biochemical and contain a GSH binding site. Medical Aspects, Coenzymes and Cofactors, eds. Dolphin, D., Glutathione is a major intracellular thiol compound of plant Poulson, R. & Avramovic, O. (Wiley, New York), Vol. 3, Part B, cells, so it could serve as the natural hydrogen donor for APS pp. 551–570. reductase. The glutathione concentration in plastids, where 10. Nikkola, M., Gleason, F. K., Saarinen, M., Joelson, T., Bjornberg, APS reductase is localized, has been variously reported be- O. & Eklund, H. (1991) J. Biol. Chem. 266, 16105–16112. tween 3 and 10 mM, and this compound is maintained 11. Tsang, M. L.-S. & Schiff, J. A. (1978) Plant Sci. Lett. 11, 177–183. predominantly in the reduced state (33, 34). Thus the apparent 12. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Lab. Press, Plainview, NY). Km[GSH] of APS reductase reported here and in reference 6 (Ϸ1 mM) is below the physiological concentration. ␥-EC is a 13. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular minor thiol compound in plants, so it could not serve as a Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, proton source for APS reductase. Significantly, GSH was Plainview, NY), 2nd Ed. 14. Laemmli, U. K. (1970) Nature (London) 227, 680–685. reported to be the most likely substrate for APS sulfotrans- 15. Segel, I. H. (1976) Biochemical Calculations (Wiley, New York), ferase (11). The experiments reported here do not specifically 2nd Ed., Appendix II. rule out the possibility that another factor such as chloroplast 16. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. thioredoxin reductase could serve to reduce APS reductase. 17. Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) However, this study and that reported in ref. 6 have established J. Bacteriol. 177, 4121–4130. that APS reductase, in the presence of physiological concen- 18. Bushweller, J. H., Åslund, F., Wu¨thrich, K. & Holmgren, A. trations of GSH, can function without such accessory factors. (1992) Biochemistry 31, 9288–9293. This finding is in agreement with the early work showing that 19. Daws, T., Lim, C.-J. & Fuchs, J. (1989) J. Bacteriol. 171, 5218– ATP and GSH are the only required factors for the reduction 5221. of sulfate to sulfite in plant chloroplasts (35). 20. Greenberg, J. T. & Demple, B. (1986) J. Bacteriol. 168, 1026– The finding that the separated APS reductase domains can 1029. catalyze APS reduction prompts the idea that this enzyme 21. Russel, M. & Model, P. (1986) in Thioredoxin and Glutaredoxin could be post-translationally processed into two proteins. We Systems: Structure and Function, eds. Holmgren, A., Bra¨nde´n, do not think this is the case because antibodies against purified C.-I., Jo¨rnvall, H. & Sjo¨berg, B.-M. (Raven, New York), pp. APR1p react with a protein from A. thaliana leaves of Ϸ48 kDa 331–337. on immunoblots (not shown). This is close to the expected 22. Boyd, D., Manoil, C. & Beckwith, J. (1987) Proc. Natl. Acad. Sci. 84, molecular mass of the protein after transport into plastids. USA 8525–8529. 23. Fahey, R. C. & Newton, G. L.. (1987) Methods Enzymol. 143, With the cloning of APS reductase from higher plants (4) 85–96. and the work reported here, long-standing questions about the 24. Brooks, S. P. J. (1992) BioTechniques 13, 906–911. nature of sulfate reduction in plants have been resolved. First, 25. Holmgren, A. (1979) J. Biol. Chem. 254, 3664–3671. the data confirm the early work on an APS-dependent enzyme 26. Tru¨mper, S., Follmann, H. & Ha¨berlein,I. (1994) FEBS Lett. 352, that is able to function with only reduced thiol compounds as 159–162. a hydrogen donor (36). Although recently purified APS sul- 27. Holmgren, A. (1979) J. Biol. Chem. 254, 9267–9632. fotransferase (6) has not yet been directly confirmed to be 28. Holmgren, A. (1979) J. Biol. Chem. 254, 3672–3678. identical to APS reductase, the similarities of these enzymes 29. Åslund, F., Nordstrand, K., Berndt, K. D., Nikkola, M., Bergman, make this highly likely. The present work also provides a T., Ponstingl, H., Jo¨rnvall, H., Otting, G. & Holmgren, A. (1996) mechanistic basis for the ability of the enzyme to use GSH J. Biol. Chem. 271, 6736–6745. directly for APS reduction, namely that APS reductase carries 30. Mao, S.-S., Holler, T. P., Yu, G. X., Bollinger, J. M., Jr., Booker, its own cofactor domain that functions as a glutaredoxin. S., Johnston, M. I. & Stubbe, J. (1992) Biochemistry 31, 9733– 9743. We thank Drs. Bruce Demple, James Fuchs, Peter Model, and 31. Wu¨rfel, M., Ha¨berlein,I. & Follmann, H. (1990) FEBS Lett. 268, Marjorie Russel for providing bacterial strains and Drs. JoAnne 146–148. Stubbe and Charles Williams for providing purified proteins. This 32. Bushweller, J. H., Billeter, M., Holmgren, A. & Wu¨thrich, K. work was funded by National Science Foundation Grant IBN-9601145. (1994) J. Mol. Biol. 235, 1585–1597. F.Å. is supported by a fellowship from the European Molecular 33. Anderson, J. W., Foyer, C. H. & Walker, D. A. (1983) Biochim. Biology Organization. Biophys. Acta 724, 69–74. 34. Foyer, C. H. & Halliwell, B. (1976) Planta 133, 21–25. 1. Schiff, J. A. (1983) in Encyclopedia of Plant Physiology, eds. 35. Schmidt, A. & Trebst, A. (1969) Biochim. Biophys. Acta 180, La¨uchli, A. & Bieleski, R. L. (Springer, Heidelberg), Vol. 15A, 529–535. pp. 401–421. 36. Schmidt, A. (1972) Arch. Microbiol. 84, 77–86. Downloaded by guest on October 1, 2021