Alternative split sites for fragment complementation,and glyphosate function as extra ligand and stabilizer for the AroA enzyme complexes

Protein reconstitution analysis can be useful for studies on protein evolution, protein folding and macromolecular assembly. AroA is a key enzyme in the pathway toward the synthesis of aromatic amino acids in microorganisms and plants, and is the target of the herbicide glyphosate. Our previous study showed that functional AroA enzyme from Escherichia coli could be reconstituted from two ~220-amino acid fragments of the protein. In this study, we explored this fragment complementation of AroA. Through a systematic study of fragment complementation, we show that successful fragment complementation can be achieved in vivo, when the split sites are within secondary structure elements as well as at loops between structure elements. In addition, we provide evidence, for the first time, that extra ligand, such as glyphosate, can function as a stabilizer of the reconstituted complexes in vivo. Therefore, our results may provide important implications for protein evolution and complex assemblies.