title: Sulfate reduction in higher plants: molecular evidence for a novel 5'-adenylylsulfate reductase.
abstract: Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5'-phosphosulfate (APS) (5'-adenylysulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. 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. 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. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5'-adenosinemonophosphate (5'-AMP). 2'-AMP, or 3'-phosphoadenosine-5'-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. 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. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.
authors.affiliation: Department of Plant Science, Rutgers University, New Brunswick, NJ 08903-0231, USA.
authors.forename: A
authors.initials: A
authors.lastname: Setya
authors.forename: M
authors.initials: M
authors.lastname: Murillo
authors.forename: T
authors.initials: T
authors.lastname: Leustek
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D013431:Sulfates; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase; C522406:APR2 protein, Arabidopsis; C019618:adenylylsulfate reductase