Identification of Bioproducts Generated Enzymatically by Cell-Free Extracts ofSaccharomyces cerevisiaefrom Cells Grown in the Presence or Absence of Heteranthin

Abstract

Ditaxis heterantha seeds are a source of the apocarotenoid heteranthin (methyl 3-oxo-12´-apo-ε-caroten-12´-oate). Saccharomyces cerevisiae can degrade, utilize this apocarotenoid, and produce seven aromas (3-oxo-α-ionone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionone, 3-hydroxy-β-cyclocitral, safranal, 4-oxo-isophorone, and isophorone). However, nothing is known about the enzymology of this process or the steps to produce them. In the present work, cell-free extracts of S. cerevisiae from cells grown in the presence or absence of heteranthin were evaluated for its degradation. When grown in heteranthin, cell-free extracts produced the same seven aromas, while in its absence only four aromas (3-hydroxy-β-cyclocitral, safranal, 4-oxo-isophorone, and isophorone) were detected. Polyacrylamide gel electrophoresis (PAGE) of cell extracts previously grown in heteranthin yielded a protein band of 27.6 kDa able to cleave this apocarotenoid. Band intensity was proportional to the heteranthin concentration and was not present in extracts from cultures grown in the absence of heteranthin. A purified sample of 27.6 kDa protein produced 3-oxo-α-ionone from heteranthin as the only reaction product, suggesting oxidation of the apocarotenoid at the 9-10 position of its moiety. Maximal enzymatic production of 3-oxo-α-ionone was observed at pH 5.5 and 35 • C with activation energy of 7.23 kJ mol −1 . The enzyme exhibited a K m value of 27 µM for heteranthin and a V max of 135 µmoles min −1 mg −1 . Our findings clearly supported the presence of at least two ways for heteranthin degradation in S. cerevisiae: one that is probably committed to the heteranthin-induced synthesis of 3-oxo-α-ionol, and the other, constitutive, likely committed to safranal production.