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

Abstract

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3-phosphoadenosine-5-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector YES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35 S-labeled substrate to acid-volatile 35 S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel ''APS reductase'' enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of Ϸ1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.Sulfur in its reduced form plays an important role in plant metabolism being involved in the biosynthesis of a wide range of primary and secondary S-containing metabolites. However the pathway of reductive assimilation of inorganic sulfate to sulfide in higher plants is the subject of some controversy (reviewed in ref. 1). After sulfate uptake, and its activation to adenosine 5Ј-phosphosulfate (APS) (5Ј-adenylylsulfate) by the enzyme ATP sulfurylase, it has been unclear whether APS is further metabolized by the enzyme APS sulfotransferase to ''bound-sulfite'' and then reduced to ''bound sulfide'' by a thiosulfonate reductase or whether the ''free-sulfite'' pathway, found in most enterobacteria and certain fungi and yeast, operates. In this latter pathway APS is converted to 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) (3Ј-phosphoadenylylsulfate) by APS kinase and PAPS is reduced to free sulfite by PAPS reductase. Sulfite is then reduced to sulfide by sulfite reductase. Discovery of a thioredoxin-dependent PAPS reductase activity in spinach (2) and the more recent suggestion that APS sulfotransferase activity is a side-reaction of APS kinase (3) provide strong support for the notion that the ''free-intermediate'' pathway operates not only in...

Regulation of sulfur assimilation in higher plants: A sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana

“…The mRNA expression of ATP sulfurylase and APS kinase was down-regulated by sulfate starvation in leaves. Expression of the halotorerant gene SAL1, which was characterized to be involved in dephosphorylation of 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to APS (22), was also depressed in sulfate-starved leaves. From these results, it was indicated that the expression of the genes involved in sulfate assimilation is not coordinately regulated by sulfate starvation in plants.…”

“…T21459) were not affected by sulfate deprivation in leaves. APR1 (21,22), recently identified to encode an APS reductase isoform responsible for the reduction of adenosine 5Ј-phosphosulfate (APS) to sulfite, increased ca. 5.5-fold in roots and 2-fold in leaves.…”

“…Recently, Setya et al (21) and Gutierrez-Marcos et al (22) demonstrated the existence of APS reductase in the higher plant A. thaliana. The existence of a PAPS-dependent sulfate-reduction pathway (41) remains unresolved.…”

Thioredoxins: structure and function in plant cells

“…Two functional thioredoxin-like molecules of 14 and 16 kDa, one of them very similar to thioredoxin h, have also been isolated from the C. reinhardtii outer dynein arm (a flagellar protein complex) (Patel-King et al, 1996). Two very recent reports also indicate the presence of a thioredoxin/glutaredoxin moiety in the sequence of three different chloroplastic PAPS reductases (or APS reductase), the thioredoxin domain being also situated in the C-terminal part of the protein (Guttierez-Marcos et al, 1996;Selyat, Murillo & Leustek, 1996). This type of organization is unique to the plant enzymes, since in bacteria and cyanobacteria PAPS reductase uses exogenous thioredoxin as an electron donor instead (Niehaus, Gisselmann & Schwenn, 1992).…”

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX

“…Members of the Trx family have a distinct Trx motif, whereas the Trx-like protein family has a second functional domain in addition to the Trx motif (9). Although Ͼ20 different types of Trx genes have been identified in the Arabidopsis genome, there are only a few Trx-like proteins, including the PKC-interacting cousin of Trx, glutaredoxin-related proteins, and protein disulfide isomerase (PDI), whose functions remain to be elucidated (10)(11)(12)(13). Among the proteins containing the Trx fold, 2-Cys peroxiredoxins (Prxs) have been shown to alter their conformation from low molecular weight (LMW) to high molecular weight (HMW) complexes in response to heat shock and radical stresses (14,15).…”