REDEFINING REDUCTIVE SULFATE ASSIMILATION IN HIGHER-PLANTS - A ROLE FOR APS REDUCTASE, A NEW MEMBER OF THE THIOREDOXIN SUPERFAMILY

The reaction steps leading from the intermediate adenosine 5'-phosphos ulfate (APS) to sulfide within the higher plant reductive sulfate assi milation pathway are the subject of controversy. Two pathways have bee n proposed: a 'bound intermediate' pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecul e to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a 'free intermediate' pa thway in which APS is further activated to 3'-phosphoadenosine 5'-phos phosulfate (PAPS) by APS kinase followed by reduction of the sulfo gro up to free sulfite by PAPS reductase. Sulfite is then reduced to foe s ulfide by sulfite reductase. Sulfide, either free or bound, is then in corporated into organic form (as cysteine) by the enzyme O-acetylserin e (thiol) lyase. In order to better characterize the pathway we attemp ted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy. We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily. We show here that the thioredoxin domain of these proteins is functional. We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to s ulfite by APS reductase and the subsequent reduction of sulfite to sul fide by sulfite reductase. In this scheme the product of the APS kinas e reaction, PAPS, is not a direct intermediate in the pathway but rath er acts as a substrate fur sulfotransferase action and perhaps as a st ore of activated sulfate that can be returned to the pathway as APS vi a phosphohydrolase action on PAPS. Interactions between enzyme isoform s within the chloroplast stroma may bring about substrate channeling o f APS and contribute to the partitioning of APS between sulfotransfera se reactions an the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the oth er. (C) 1998 Elsevier Science ireland Ltd. All rights reserved.