A novel bifunctional fusion enzyme catalyzing ethylene synthesis via 1-aminocyclopropane1-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 heterologous prokaryotic Escherichia coli system, which is capable of converting endogenous S-adenosyl-L-methionine (AdoMet) to ethylene. The molecular weight of the fusion enzyme, ACSO, as determined by sodium dodecyl sulfate-polyacrylamide 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 in ethylene production catalyzed by the bifunctional ACSO was accompanied by a decrease in ACC accumulation. Similarly, in E. coli cells, the level of ACC, produced as an intermediate during the sequential reactions from AdoMet to ethylene, was also found to arise earlier than that 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 bifunctional fusion enzyme to be useful as a reporter and for research in molecular biology, developmental biology, fermentation, and genetic engineering.
Li
N
N
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
Jiang
X N
XN
Cai
G P
GP
Yang
S F
SF
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000598:Amino Acids, Cyclic; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005030:Ethylenes / Q000096:biosynthesis*; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D018551:Solanum lycopersicum; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013025:Glycine max
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D005030:Ethylenes; D011993:Recombinant Fusion Proteins; C023863:1-aminocyclopropane-1-carboxylic acid; D012436:S-Adenosylmethionine; C036216:ethylene; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase