Reconstitution of the enzyme AroA and its glyphosate tolerance by fragment complementation.
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 aroA 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.
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China.
Yi-Cheng
YC
Sun
Yan
Y
Li
Hai
H
Zhang
Hai-Qin
HQ
Yan
David N
DN
Dowling
Yi-Ping
YP
Wang
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000737:chemistry / Q000502:physiology*; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004361:Drug Tolerance; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology; D016958:Pseudomonas putida / Q000201:enzymology; D000097797:Glyphosate
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D001426:Bacterial Proteins; D005998:Glycine; D006540:Herbicides; D010446:Peptide Fragments; D000097797:Glyphosate