Open AccessExpression and characterization of three tomato 1‐aminocyclopropane‐1‐carboxylate oxidase cDNAs in yeast
Author(s) -
Bidonde Sylvie,
Ferrer Maria Angeles,
Zegzouti Hicham,
Ramassamy Sabine,
Latché Alain,
Pech JeanClaude,
Hamilton Andrew J.,
Grierson Donald,
Bouzayen Mondher
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2530020.x
Subject(s) - yeast , biochemistry , saccharomyces cerevisiae , complementary dna , enzyme , heterologous expression , in vivo , chemistry , oxidase test , isozyme , microbiology and biotechnology , biology , gene , recombinant dna , genetics
Heterologous expression in yeast has previously shown that the tomato cDNA LE‐ACO1 encodes a functional 1‐aminocyclopropane‐1‐carboxylate (ACC) 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 family ( LE‐ACO2 and LE‐ACO3 ) were isolated and expressed in Saccharomyces cerevisiae . Analysis of the predicted amino acid sequences showed that 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 in vivo . ACO activities assayed in vitro revealed no significant differences between the three isoforms with regards to optimum temperature (29 °C), optimum pH (6.8−7.2), absolute dependence for ascorbate, Fe 2+ and carbon dioxide, and inhibition by iron‐chelating agents (1,10‐phenanthroline and EDTA), Co 2+ and free‐radical scavengers ( n ‐propyl gallate). However, differences were detected in the apparent K m values for ACC, the pI and the specific activity. The biochemical features that might explain the differences between the isoenzyme activities are discussed.