EXPRESSION AND CHARACTERIZATION OF 3 TOMATO 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE CDNA IN YEAST

Heterologous expression in yeast has previously shown that the tomato cDNA LE-ACO1 encodes a functional 1-aminocyclopropane-1-carboxylate (A CC) oxidase (ACO) protein [Hamilton, A. J., Bouzayen, M. & Grierson, D . (1991) Proc. Natl Acad. Sci. USA 88, 7434-7437]. In the present work , full-length cDNAs encoding the two other members of the tomato ACO f amily (LE-ACO2 and LE-ACO3) were isolated and expressed in Saccharomyc es cerevisiae. Analysis of the predicted amino acid sequences showed t hat the ACO1 and ACO3 proteins are highly similar (95%) while ACO2 is more divergent (89%). Yeast strains transformed with each of the three cDNAs were able to convert exogenous ACC to ethylene, the ACO1 strain exhibiting the highest activity in vivo and the ACO3 and ACO2 strains reaching 65% and 45% of ACO1 maximum activity, respectively. None of the ACO activities expressed in yeast required addition of ascorbate i n vivo. ACO activities assayed in vitro revealed no significant differ ences between the three isoforms with regards to optimum temperature ( 29 degrees C), optimum pH (6.8 - 7.2), absolute dependence for ascorba te, Fe2+ and carbon dioxide, and inhibition by iron-chelating agents ( 1,10-phenanthroline and EDTA), Co2+ and free-radical scavengers (n-pro pyl gallate). However differences were detected in the apparent K-m va lues for ACC, the pI and the specific activity. The biochemical featur es that might explain the differences between the isoenzyme activities art discussed.