Two pathways in the biosynthesis of cadystins (γEC)nG in the cell-free system of the fission yeast

Abstract

Small metal-binding peptides, cadystins, with the general structure of (γ-Glu-Cys)_n-Gly ((γEC)_nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120 000 × g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is γ-Glu-Cys (γEC) dipeptidyl transfer from both glutathione (γECG) and cadystins to glutathione and cadystins. The other one is γEC polymerization from (γEC)_n and glutathione to (γEC)_n+i, followed by glycine addition with glutathione synthetase.Key words: cadystin, fission yeast, cell-free biosynthesis, dipeptidyl transferase.