Abstract
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.
Highlights
Protein reconstitution analysis can be useful for studies on protein evolution, protein folding and macromolecular assembly
5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA) is a key enzyme involved in aromatic amino acid biosynthesis in bacteria, fungi, and higher plants, and is the target of the global herbicide glyphosate [9,10]
In our study we found that AroA activity reconstitution could be achieved, when the split sites are located at loops between folding units, or within secondary structure elements, or at loops between secondary structure elements
Summary
Protein reconstitution analysis can be useful for studies on protein evolution, protein folding and macromolecular assembly. Our previous study showed that functional AroA enzyme from Escherichia coli could be reconstituted from two ~220-amino acid fragments of the protein. 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA) is a key enzyme involved in aromatic amino acid biosynthesis in bacteria, fungi, and higher plants, and is the target of the global herbicide glyphosate [9,10]. Our previous study showed that functional AroA could be reconstituted from two fragments of the protein corresponding to a.a. 1−218 and 219−427 [16]. These fragments result from a split site located in the loop between two β strands (Table 1). To test whether this split site is unique for fragment complementation of AroA, we systematically
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