ABSTRACT1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KE-FAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.The simplest olefin, ethylene, is a gaseous plant hormone which regulates many physiological processes of plant growth and development. The biosynthetic pathway for ethylene in higher plants has been elucidated as follows:. Two enzymes that are unique to this pathway are ACC synthase and ACC oxidase (also known as ethylene-forming enzyme). ACC synthase converts S-adenosylmethionine to ACC, and ACC oxidase catalyzes the oxidation of ACC to ethylene. In fruits, both ACC synthase and ACC oxidase are induced during ripening and contribute to the regulation of ethylene biosynthesis.While much progress has been achieved on the characterization of ACC synthase at the biochemical and molecular levels (1-5), progress on the isolation of in vitro ACC oxidase has been slow. ACC oxidase activity is readily demonstrated in vivo by supplying tissues with ACC. These in vivo studies have established that the enzyme has a high affinity for its substrate ACC, exhibits stereospecificity toward the stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid for the synthesis of 1-butene, and specifically converts ACC to ethylene, CO2, and cyanide (6). Although a number of plant enzyme preparations were shown to be capable of oxidizing ACC to ethylene, these preparations lacked these characteristics. Based on these criteria it was concluded that these reported in vitro systems were not the authentic ACC oxidase that functions in vivo (6).Recent advances in the molecular biology of this enzyme have led to the successful isolation ofauthentic ACC oxidase. Based on the observations that ACC oxidase activity in tomato fruit was greatly reduced by an antisense gene of a ripening-related cDNA, pTOM13, Hamilton et al. (7) suggested that the pTOM13 gene was related to ACC oxidase. Later work confirmed that pTOM13 and relat...

Ethylene-forming enzyme (EFE) was isolated from apple (Malus domestica Borkh. cv Golden Delicious) fruit tissue. The enzyme activity in the homogenate is associated with the pellet fraction and can be solubilized with Triton X-100 or polyvinylpolypyrrolidone. The solubilized enzyme system resembles the in vivo system in that it exhibits a low Km (17 micromolar) for its substrate 1-aminocyclopropane-1-carboxylic acid (ACC), is stereospecific toward 2-ethyl-ACC stereoisomers for 1-butene production, and is inhibited by cobalt ions and a-aminoisobutyric acid. Intact preclimacteric fruits treated with exogenous ethylene showed a marked increase in in vivo EFE activity and this increase was accompanied by a parallel increase in in vitro EFE activity. These results support the notion that the isolated EFE represents the authentic in vivo activity.Ethylene is a plant hormone that is involved in a number of physiological processes such as fruit ripening and plant senescence (1). It is biosynthesized in higher plants via the following pathway: methionine --S-adenosylmethione --ACC2 -* ethylene (2). The final step is catalyzed by the EFE, which has been well characterized in vivo (18). Study of reaction products reveals that EFE catalyzes the following reaction (12): ACC + ½2 02 --C2H4 + HCN + CO2 + H20.In vivo studies indicate that EFE has a high affinity for ACC and displays stereospecificity toward stereoisomers of AEC from which 1-butene is produced (7,11,16). Much work has been carried out in isolating the enzyme that catalyzes the oxidation of ACC to ethylene, and a number of cellfree, ethylene-forming systems have been described (for a review see ref. 18). Although these systems are dependent on oxygen, except for intact protoplasts and vacuoles that possess native EFE properties (4), they lack high affinity for ACC and display no stereospecificity for AEC stereoisomers (11,16). Although progress on the isolation of cell-free EFE has been slow, important advances in the molecular biology of this 'This work was supported by grant DCM-9004129 from the National Science Fundation. J.C.F-M. was a recipient of a research fellowship awarded by Ministerio de Educaci6n y Ciencia, Spain.2 Abbreviations: ACC, 1 -aminocyclopropane-1 -carboxylic acid; AEC, 1-amino-2-ethylcyclopropane-1 -carboxylic acid; AIB, a-aminoisobutyric acid; EFE, ethylene-forming enzyme; PVPP, polyvinylpolypyrrolidone.enzyme have been made recently. Based on the observations that EFE activity in tomato fruit was greatly reduced with a pTOM 13 antisense gene, Hamilton et al. (6) suggested that the pTOM 13 gene product is related to EFE. Later work confirmed that pTOM 13 confers EFE activity when expressed in yeast (5) or Xenopus oocytes (15). The deduced amino acid sequence of pTOM 13 shows homology with that of a flavanone-3-hydroxylase. Although Yang and Hoffman (18) suggested as early as 1984 that EFE may be an ACC hydroxylase, attempts to isolate and assay EFE as a hydroxylase have not been seriously pursued until recently. Ververidis and Joh...

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