A Novel Bifunctional Fusion Enzyme Catalyzing Ethylene Synthesis via 1-Aminocyclopropane1-carboxylic Acid

Ning Li,Xiang Ning Jiang,Guo Ping Cai,Shang Fa Yang

doi:10.1074/jbc.271.42.25738

Ning Li, Xiang Ning Jiang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.42.25738

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Abstract

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.

Highlights

Sions, inhibiting seedling elongation, stimulating root initiation, promoting initiation of the flowering of pineapples, and increasing latex flow in rubber trees
A 54-nucleotide DNA fragment was cleaved from the 3Ј end of the ACC synthase cDNA, which resulted in the deletion of 18 aa from the C terminus of ACC synthase
WI) (Fig. 1A), pretreated with NdeI and BamHI. This insertion created a fusion of a short polypeptide with an array of 6 histidines, His-tag [34], attached to the N terminus of the hybrid enzyme created by the first fusion of soybean ACC synthase and tomato ACC oxidase (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology Reagents and Methods—The primers, PACS1 (5ЈGAC C ATATG GGG TTG ATG GAT GTG G-3Ј) and PACS2 (5ЈAAA GGATCC TCA AAT TGT GGC TTT AAC CAG AGG-3Ј), corresponding to nucleotides 157–181 and 1594 –1618 of soybean ACC synthase cDNA (GMCACCS1; accession number X67100; Ref. 26), respectively, and two additional primers, PACO1 (5Ј-ATT C AT ATG GAT AT C TTC CCA ATT ATT AAC TTG-3Ј) and PACO2 (5Ј-CGA GGATCC TTA AGC ACT TGC AAT TTG ATC A-3Ј), corresponding to nucleotides 166 –198 and 1098 – 1119 of tomato ACC oxidase cDNA (LEETHYBR or pCR13; accession number X04792; Ref. 17), respectively, were synthesized using an automated DNA synthesizer, Gene Assembler (Pharmacia). An EcoRV site (underlined) was introduced into the PACO1 primer downstream of the NdeI site only for fusion of the N terminus of ACC oxidase to the C terminus of ACC synthase. Both soybean plant and tomato fruit cDNA libraries used in this experiment were made by Stratagene. Assay for the Bifunctional Enzyme Activities—The ACC synthase and ACC oxidase activity assays were performed according to the methods described previously [7,8,9,10, 33]. The reaction was performed at 30 °C for 30 min with constant shaking in a 4-ml test tube, which contained, in a total volume of 0.5 ml, 20 mM Tris-HCl (pH 7.6), 1 mM dithiothreitol, 20 ␮M PLP, 30 mM sodium ascorbate, 20 mM sodium bicarbonate, 20 ␮M ferrous sulfate, and 20% glycerol

RESULTS

DISCUSSION

Methods