A NOVEL BIFUNCTIONAL FUSION ENZYME CATALYZING ETHYLENE SYNTHESIS VIA 1-AMINOCYCLOPROPANE 1-CARBOXYLIC ACID

A C terminus truncated soybean 1-aminocyclopropane-1-carboxylic acid ( ACC) synthase (466 aa) was fused to an N terminus truncated tomato ACC oxidase (312 aa) to create a 778-amino acid fusion polypeptide, This ACC synthase-ACC oxidase fusion enzyme (ACSO) was expressed in a heter ologous prokaryotic Escherichia coli system, which is capable of conve rting endogenous S-adenosyl-L-methionine (AdoMet) to ethylene, The mol ecular weight of the fusion enzyme, ACSO, as determined by sodium dode cyl sulfate-polyacryl amide gel electrophoresis, was 90 +/- 3 kDa. Gel filtration analysis indicates that the native ACSO is oligomeric and is capable of converting exogenously supplied AdoMet to ethylene, The ethylene production rate of ACSO fusion enzyme was determined to be 6. 0 nmol h(-1) mg(-1) under our assaying conditions using the partially purified enzyme extract, In the enzyme reaction mixture, an increase i n ethylene production catalyzed by the bifunctional ACSO was accompani ed by a decrease in ACC accumulation. Similarly, in E. coli cells, the level of ACC, produced as an intermediate during the sequential react ions from AdoMet to ethylene, was also found to arise earlier than tha t of ethylene, Because ACSO could produce ethylene from the ubiquitous AdoMet in living cell and the method commonly used to measure gaseous ethylene is simple, fast, and sensitive, we anticipate this bifunctio nal fusion enzyme to be useful as a reporter and for research in molec ular biology, developmental biology, fermentation, and genetic enginee ring.