Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

Abstract

Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis, i.e. methylation of homocysteine. A search of theSchizosaccharomyces pombegenomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, and thus the gene was identified as a methionine synthase and designatedmet26. Themet26mutant was found to exhibit a remarkable growth defect in the absence of adenine even in medium supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeastSaccharomyces cerevisiae, which has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes fromSac. cerevisiaeconstituting the cystathionine pathway (CYS4andCYS3) intoSch. pombeΔmet26cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in Δmet26cells was higher than in other methionine auxotrophs and that introduction of theSac. cerevisiaecystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in themet26mutant.