Gene-enzyme relationship in the sulfate assimilation pathway of Saccharomyces cerevisiae. Study of the 3'-phosphoadenylylsulfate reductase structural gene.
In yeast, mutations in six different loci (MET1, MET4, MET8, MET16, MET22, and MET25) have been reported to result in the absence of 3'-phosphoadenylylsulfate (PAPS) reductase activity. In the present study, we show that MET16 is the structural gene for PAPS reductase and that the yeast and the Escherichia coli enzymes display significant similarities. Thioredoxin has been implicated in the reduction of PAPS in Saccharomyces cerevisiae as well as in E. coli. One of the generally accepted mechanisms for the action of thioredoxin as a hydrogen donor involves a redox-active sulfhydryl group in the catalytic site of PAPS reductase. However, the present study shows that the site-directed mutagenesis of the unique cysteine from PAPS reductase leads to an enzyme which remains active in vivo. This result would rather support the hypothesis of thioredoxin playing the role of a thiol carrier in the reduction of PAPS into sulfite. Strains separately mutated in the six different loci cited above were examined for the expression of different genes. A mutation in the MET4 gene abolishes transcription of both genes MET16 and MET25. In contrast, mutations in MET1, MET8, and MET25 do not impair MET16 transcription, yet strains bearing these mutations are devoid of PAPS reductase activity. To account for the latter result, we postulate that the enzymes involved in sulfate assimilation may occur as a multienzyme complex in S. cerevisiae.
Thomas
D
D
Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Barbey
R
R
Surdin-Kerjan
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D005838:Genotype; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013431:Sulfates / Q000378:metabolism*
D013431:Sulfates; D010088:Oxidoreductases; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase