Sulfate assimilation in higher plants characterization of a stable intermediate in the adenosine 5'-phosphosulfate reductase reaction.
The enzyme catalysing the reduction of adenosine 5'-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 degrees C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2-3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5'-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 degrees C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2-3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2-3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.
Weber
M
M
Institute of Plant Physiology, Bern, Switzerland.
Suter
M
M
Brunold
C
C
Kopriva
S
S
D000250:Adenosine Phosphosulfate / Q000737:chemistry; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010944:Plants / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism*; D013447:Sulfites / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013879:Thioredoxins / Q000737:chemistry
D013431:Sulfates; D013447:Sulfites; D013462:Sulfur Radioisotopes; D000250:Adenosine Phosphosulfate; D013879:Thioredoxins; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase