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Reconstitution of the enzyme AroA and its glyphosate tolerance by fragment complementation
Author(s) -
Sun Yi-Cheng,
Li Yan,
Zhang Hai,
Yan Hai-Qin,
Dowling David N.,
Wang Yi-Ping
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.01.075
Subject(s) - aroa , complementation , fragment (logic) , glyphosate , enzyme , chemistry , biochemistry , biology , microbiology and biotechnology , gene , mutant , escherichia coli , computer science , enterobacteriaceae , programming language
5‐Enolpyruvylshikimate‐3‐phosphate (EPSP) synthase (AroA) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, and is the target of the herbicide glyphosate. Glyphosate tolerance activity of the enzyme could be obtained by natural occurrence or by site‐directed mutagenesis. A functional Pseudomonas putida AroA was obtained by co‐expression of two protein fragments AroA P. putida ‐N210 and AroA P. putida ‐C212 in Escherichia coli aro A mutant strain AB2829. From sequence analysis, the equivalent split site on E. coli AroA was chosen for further study. The result indicated that functional E. coli AroA could also be reconstituted from two protein fragments AroA E. coli ‐N218 and AroA E. coli ‐C219, under both in vivo and in vitro conditions. This result suggested that the fragment complementation property of this family of enzyme may be general. Additional experiments indicated that the glyphosate tolerance property of AroA could also be reconstituted in parallel with its enzyme activity. The implication of this finding is discussed.