3 MEMBERS OF A NOVEL SMALL GENE-FAMILY FROM ARABIDOPSIS-THALIANA ABLETO COMPLEMENT FUNCTIONALLY AN ESCHERICHIA-COLI MUTANT DEFECTIVE IN PAPS REDUCTASE-ACTIVITY ENCODE PROTEINS WITH A THIOREDOXIN-LIKE DOMAIN AND APS REDUCTASE-ACTIVITY

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3'-phosphoadenosine -5'-phosphosulfate (PAPS) reductase homolog], have been isolated by co mplementation of an Escherichia coli cysH mutant, defective in PAPS re ductase activity, to prototrophy with an Arabidopsis thaliana cDNA lib rary in the expression vector lambda YES. Sequence analysis of the cDN As revealed continuous open reading frames encoding polypeptides of 46 5, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fung al, yeast, and bacterial PAPS reductases. However, unlike microbial PA PS reductases, each PRH protein has an N-terminal extension, character istic of a plastid transit peptide, and a C-terminal extension that ha s amino acid and deduced three-dimensional homology to thioredoxin pro teins, Adenosine 5'-phosphosulfate (APS) was shown to be a much more e fficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert S-35-labeled substrate to acid-vol atile S-35-sulfite. We speculate that the thioredoxin-like domain is i nvolved in catalytic function, and that the PRH proteins may function as novel ''APS reductase'' enzymes. Southern hybridization analysis sh owed the presence of a small multigene family in the Arabidopsis genom e, RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of approximate to 1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowled ge, this is the first report of the cloning and characterization of pl ant genes that encode proteins with APS reductase activity and support s the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilati on.