Abstract
Three plastidic chorismate synthase isozymes (CS1, CS2 and CS2Δ) of tomato were identified by isolation of the corresponding cDNAs. These three cDNAs are derived from only two genes (LeCS1 and LeCS2). This additional complexity results from differential splicing of the primary transcript of one of the genes (LeCS2) giving rise to two different transcripts (CS2 and CS2Δ transcripts). All three isozymes were individually expressed in Escherichia coli both as precursor proteins with N-terminal transit peptides and as mature proteins. Only the mature but not the precursor isozymes CS1 and CS2 were enzymatically active. The enzyme CS2Δ was unstable in E. coli. Both CS1 and CS2 were purified to near homogeneity and their enzymatic properties were analyzed. They differ substantially in their K m values for the substrate 5-enol-pyruvylshikimate 3-phosphate (11 and 80 μM for the mature forms of CS1 and CS2, respectively). The two isozymes appear to be active only as oligomers, and the potential physiological implications of this result are discussed.
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Abbreviations
- CS:
-
chorismate synthase
- CS1, CS2, CS2Δ:
-
tomato chorismate synthase isozymes
- DMS:
-
[dimethylsuberimidate
- EPSP:
-
5-enolpyruvylshikimate 3-phosphate
- IPTG:
-
isopropylβ-d-thiogalactopyranoside
- LeCS1, LeCS2 :
-
tomato chorismate synthase genes
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Dedicated to Professor Andreas Sievers, Bonn, on the occasion of his 65th birthday
We thank Drs. J.R. Coggins (Department of Biochemistry, University of Glasgow, UK) for the E. coli strain AB2849, the plasmid pGM602 and his help in establishing the continuous CS assay, W. Staudenmann (ETH-Ziirich, Switzerland) for the determination of the N-terminal sequences and the determination of the molecular mass of MatCSl, P. Pfister (ETH-Zurich) for his help with the fermentor, D. Rubli (ETH-Zurich) for preparing the photographs, and A. Schaller (ETH-Ziirich) for critically reading the manuscript. This work was supported by grants from the Swiss National Science Foundation (to J.S. and N.A.).
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Braun, M., Henstrand, J.M., Görlach, J. et al. Enzymatic properties of chorismate synthase isozymes of tomato (Lycopersicon esculentum Mill.). Planta 200, 64–70 (1996). https://doi.org/10.1007/BF00196650
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DOI: https://doi.org/10.1007/BF00196650