Adenosine 5′-Phosphosulfate Sulfotransferase and Adenosine 5′-Phosphosulfate Reductase Are Identical Enzymes

Abstract

Adenosine 5-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M r of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K m ‫؍‬ 6.5 M) and not adenosine 3-phosphate 5-phosphosulfate as sulfonyl donor. The V max of recombinant Lemna APS sulfotransferase (40 mol min ؊1 mg protein ؊1 ) was about 10 times higher than the previously published V max of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO 3 2؊ . Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO 3 2؊ was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.Higher plants and many microorganisms growing with sulfate as sulfur source reduce it to the level of sulfide for the synthesis of cysteine, methionine, coenzymes, and iron-sulfur clusters of enzymes (1-5). The reaction sequence from sulfate to sulfide is called assimilatory sulfate reduction as opposed to dissimilatory sulfate reduction, which occurs in certain anaerobic organisms such as Desulfovibrio and Desulfotomaculum, where sulfate functions as an electron acceptor during oxidation of organic substrates and where reduced forms of sulfur are excreted into the surroundings (3).The first step of assimilatory sulfate reduction is an activation of sulfate catalyzed by ATP sulfurylase (EC 2.7.7.4). The adenosine 5Ј-phosphosulfate (APS) 1 is the substrate for APS kinase (EC 2.7.1.2.5), which forms adenosine 3Ј-phosphate 5Ј-phosphosulfate (PAPS) in a second activation step (1-5). The subsequent reduction sequence starting from PAPS is well established in bacteria (4) and fungi (6), where a PAPS reductase (EC 1.8.99) reacts first with reduced thioredoxin then with PAPS to form SO 3 2Ϫ , oxidized thioredoxin, and adenosine 3Ј-phosphate 5Ј-phosphate (PAP) (4, 7, 8). The SO 3 2Ϫ is reduced to sulfide by sulfite reductase (EC 1.8.7.1). Sulfide is finally incorporated into O-acetyl-L-serine via O-acetyl-L-serine thiollyase (EC 4.2.99.8), thus forming cysteine (1-4). All these enzymes were detected in plants (1-5, 7-11), indicating that the identical reaction sequence is operative. In two early reports it was demonstrated, however, that plants and algae use APS rather than PAPS as sulfonyl donor for the first reduction step (12, 13). APS-dependent enzymes were partially p...

The Presence of an Iron-Sulfur Cluster in Adenosine 5′-Phosphosulfate Reductase Separates Organisms Utilizing Adenosine 5′-Phosphosulfate and Phosphoadenosine 5′-Phosphosulfate for Sulfate Assimilation

“…The enzyme produces sulfite from APS but not from PAPS, and the activity is stimulated by thioredoxin m but is not absolutely dependent on this compound. The reported V max of the Pseudomonas APR, 5.8 mol min Ϫ1 mg Ϫ1 (19), is almost identical to that of the N-domain of APR from Arabidopsis, 5.1 mol min Ϫ1 mg Ϫ1 , 2 being ϳ8 times lower than the V max of recombinant APR from Lemna minor (13). tive site, exactly like the enzyme from higher plants (14).…”

“…The plant APS reductase (APR), recently cloned from Arabidopsis thaliana, is a protein composed of two distinct domains: an N-terminal part is homologous to the Escherichia coli PAPS reductase (encoded by the cysH gene), and a C-terminal part is similar to thioredoxin with a function modified toward glutaredoxin (10 -12). This enzyme is identical to the previously described APS sulfotransferase (13) and contains a [4Fe-4S] iron-sulfur cluster as a cofactor (14). APS reductase is a highly regulated enzyme, and it is considered to have a major control on the flux through assimilatory sulfate reduction in plants (3,15).…”

“…Metal foil consisting of 57 Fe (94.7% enrichment; Glaser, Basel, Switzerland) was dissolved in HCl, neutralized, and added to the culture medium at a final 57 Fe concentration of 20 M. Cloning of APS Reductase from Plectonema-DNA was isolated from 0.5 g of Plectonema strain 73110 cells according to the standard procedures (28). The major part of the APR coding region was amplified from the DNA by PCR with primers derived from regions conserved in plant APR and bacterial PAPS reductase sequences as described by Suter et al (13). The PCR product was cloned by the TA cloning kit (Invitrogen), and three independent inserts were completely sequenced on both strands.…”

Differential Subcellular Localization and Expression of ATP Sulfurylase and 5′-Adenylylsulfate Reductase during Ontogenesis of Arabidopsis Leaves Indicates That Cytosolic and Plastid Forms of ATP Sulfurylase May Have Specialized Functions

“…Two catalytically distinct forms of ATP sulfurylase have been purified from Euglena gracilis, one of which exists in mitochondria (Li et al, 1991). APS reductase, formerly termed APS sulfotransferase (Suter et al, 2000), was found to be exclusively plastid-localized (Fankhauser and Brunold, 1978;Brunold and Suter, 1989;Rü egsegger and Brunold, 1993). In Euglena an isoform is also localized in mitochondria (Saidha et al, 1988).…”

“…In contrast, APS is used directly as the substrate for the reduction pathway. Reduced glutathione-dependent APS reductase (EC 1.8.99.-) transfers two electrons to form sulfite (Suter et al, 2000). Then ferredoxin-dependent sulfite reductase completes the reduction to sulfide.…”