The first enzyme of sulfate assimilation (ATP-sulfurylase or ATP:sulfate adenylyl transferase) [EC 2.7.7.4], catalyzes the formation of adenosine-5'-phosphosulfate from ATP and sulfate. Arabidopsis thaliana contains a three-member, highly homologous, expressed gene family encoding plastid localized forms of ATP sulfurylase; APS1 (Leustek et al, 1994), APS2, and APS3 (Murillo and Leustek, 1995). All three cDNA clones functionally complement a met3 (ATP sulfurylase) mutant strain of Saccharomyces cerevisiae Leustek et al, 1994; Murillo and Leustek, 1995). APS1 is the most highly expressed member of this gene family. The APS polypeptides share homology with ATP-sulfurylases from fungi, a marine worm and a chemoautotrophic bacterium, but not from Escherichia coli or Rhizobium meliloti (Murillo and Leustek, 1995). Analysis of recombinant APS3 indicates that the protein is structurally and kinetically similar to fungal ATP-sulfurylase, but very different from the E. coli enzyme. The APS3 polypeptide is a homotetramer. Despite the sequence, structural, and kinetic differences between higher plant and E. coli ATP-sulfurylases, APS2 and APS3 are able to functionally complement E. coli cysD and cysN (ATP-sulfurylase) mutant strains (Murillo and Leustek, 1995).

The ATP sulfurylase gene (APS2) of Arabidopsis thaliana has been overexpressed in tobacco cell cultures (Hatzfeld et al, 1998). ATP sulfurylase activity was elevated 8-fold, but had no effect on ³⁵S-sulfate uptake or ATP sulfurylase regulation, except that ATP sulfurylase activity variations in response to S starvation were 8 times higher than wildtype. Overexpression of the Arabidopsis APS2 gene in tobacco did not confer resistance to the sulfate analog, selenate (Hatzfeld et al, 1998). However, overexpression of the APS1 gene (encoding a plastidic ATP sulfurylase) in Indian mustard confers increased selenate uptake, reduction and tolerance (Pilon-Smits et al, 1999). The depression of ATP sulfurylase by S deficiency may involve a posttranscriptional control mechanism (Hatzfeld et al, 1998).

A single locus encodes 5'-adenylylsulfate (APS) kinase [EC 2.7.1.25] (APK) in Arabidopsis thaliana (Lee and Leustek, 1998). APK mRNA is slightly more abundant in leaves than in roots and its level does not change in response to sulfur starvation. The APK protein, synthesized in vitro, is able to enter isolated intact chloroplasts (Lee and Leustek, 1998).

A cDNA from Arabidopsis thaliana encoding the APS-kinase [EC 2.7.1.25] was modified by deletion of a plastidic transit peptide to enable its expression in Escherichia coli (Schiffmann and Schwenn, 1994). The resultant protein was enzymatically active as APS-kinase and restored prototrophic growth in an APS-kinase mutant. Moreover all transformants with the modified plant DNA also acquired APS-sulfotransferase activity. Monospecific polyclonal antibodies raised against the APS-kinase as immunogen also reacted against APS-sulfotransferase. It is therefore proposed that APS-sulfotransferase activity is a nonphysiological side reaction of APS-kinase (Schiffmann and Schwenn, 1994).

The pathway by which plants reduce activated sulfate to sulfite has been the subject of considerable debate. Two leading hypotheses for the mechanism of sulfate reduction in higher plants are: 1. adenosine 5'-phosphosulfate (APS) (5'-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione (this may involve a 'bound intermediate' pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate which is then further reduced by thiosulfonate reductase to bound sulfide (Wray et al, 1998)); 2. the mechanism may be similar to that in bacteria in which 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase catalyzes thioredoxin (Trx)-dependent reduction of PAPS (Setya et al, 1996; Wray et al, 1998). Recent discoveries on two enzymes (APS sulfotransferase and APS reductase) have clarified this issue (Bick and Leustek, 1998; Wray et al, 1998). It is now proposed that these proteins are the same enzyme and that the sulfate assimilation pathway in plants differs from that in other sulfate assimilating organisms (Bick and Leustek, 1998; Leustek and Saito, 1999; Leustek et al, 2000). As shown in red in the metabolic scheme above, the main pathway of sulfate reduction in plants appears to be via APS rather than PAPS. Thus, three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, APR-2, and APR-3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli (Setya et al, 1996; Gutierrez-Marcos et al, 1996; Wray et al, 1998). The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. However, further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate (Setya et al, 1996; Gutierrez-Marcos et al, 1996). The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2 (Setya et al, 1996). Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members (Setya et al, 1996).

The 5'-adenylylsulfate (APS) reductase [EC 1.8.99.-] of plants has a glutathione (GSH)-dependent reductase domain that functions similarly to the redox cofactor glutaredoxin (Bick et al, 1998). The APR1 cDNA encoding APS reductase from Arabidopsis thaliana is able to complement the cysteine auxotrophy of the Escherichia coli cysH mutant, only if the E. coli strain produces glutathione. The purified recombinant enzyme (APR1p) can use GSH efficiently as a hydrogen donor in vitro, showing a K_m of approx. 0.6 mM for GSH. The R domain of the protein is homologous with microbial PAPS reductase, the C domain is homologous with thioredoxin. The C domain can substitute for glutaredoxin in vivo as demonstrated by complementation of an E. coli mutant (Bick et al, 1998).

A cDNA encoding a plant-type APS reductase was isolated from a cell suspension culture of Catharanthus roseus. The cDNA encodes a protein consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase (Prior et al, 1999). The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa (Prior et al, 1999). Deletions lacking the transit sequence liberated sulfite from APS in vitro with glutathione as reductant. A deletion lacking the transit sequence and C-terminal domain required exogenous thioredoxin as reductant (Prior et al, 1999).

In contrast to plants, the adenylylsulfate reductase [EC 1.8.99.2] of Desulfovibrio vulgaris catalyzes electron transfer from dihydroflavin coenzyme (FADH₂, FMNH₂, or dihydroriboflavin) to APS, and catalyzes flavin-mediated oxidation of ferrocytochrome c3 with APS (Yagi and Ogata, 1996).

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Gutierrez-Marcos JF, Roberts MA, Campbell EI, Wray JL 1996 Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and "APS reductase" activity. Proc. Natl. Acad. Sci. U.S.A. 93: 13377-13382.

Hatzfeld Y, Cathala N, Grignon C, Davidian JC 1998 Effect of ATP sulfurylase overexpression in bight yellow 2 tobacco cells. Regulation of ATP sulfurylase and SO₄^2- transport activities. Plant Physiol. 116: 1307-1313.

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Pilon-Smits EAH, Hwang S, Lytle CM, Zhu Y, Tai JC, Bravo RC, Chen Y, Leustek T, Terry N 1999 Overexpression of ATP sulfurylase in Indian mustard leads to increased selenate uptake, reduction, and tolerance. Plant Physiol. 119: 123-132.

Prior A, Uhrig JF, Heins L, Wiesmann A, Lillig CH, Stoltze C, Soll J, Schwenn JD 1999 Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures. Biochim. Biophys. Acta 1430: 25-38.

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