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 reductase 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 enzyme is a glutathione (GSH)-dependent reductase that whereas the reductase may produce sulfite (4), although the functions similarly to the redox cofactor glutaredoxin. 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 thioredoxinyglutaredoxin 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 ' 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 thioredoxin and glutaredoxin. This structure suggests that 73–327 (the R domain), homologous with microbial PAPS a dithiol–disulfide 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- Proteins 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- ' of 1 mM. The R domain does not show these activities. The mation as electrons are transferred to a variety of reductases C domain is active in GSH-dependent reduction of insulin (3). Thioredoxin is specifically reduced by NADPH or ferre- disulfides and ribonucleotide reductase, whereas APR1p and doxin-dependent thioredoxin reductase (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 glutathione reductase (9). The specificity for GSH between the two enzymes. lies in a glutathione binding site 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 59-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- 39-phosphoadenosine-59-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 prokaryotes 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 Arabidopsis thaliana (4, glutathione reductase (G4759) were from Sigma. Bovine thi- 5). This enzyme was termed a ‘‘reductase’’ because of its homology with the protein 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); g g 9 9 The publication costs of this article were defrayed in part by page charge -EC, -glutamylcysteine; APS, 5 -adenylylsulfate; PAPS, 3 - phosphoadenosine-59-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 0027-8424y98y958404-6$2.00y0 Dudley Road, Rutgers University, New Brunswick, NJ 08901-8250. PNAS is available online at http:yywww.pnas.org. e-mail: [email protected]. 8404 Downloaded by guest on September 28, 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 P1kc1 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 D(ara–leu)7697 (22) from the difference in absorbance at 280 and 260 nm (15), and FA87 DtrxA, trxC, grxA::km † the concentrations of proteins in crude extracts were measured pBAD39-trxC by the Bradford method (16). D(ara–leu)7697 Construction of Expression Plasmids and Purification of FA47 DtrxA, grxA::km † Recombinant Proteins. For protein purification the Novagen D(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 '400-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% (wtyvol) 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.