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KMID : 0545120070170081385
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 8 p.1385 ~ p.1389
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Engineering and Characterization of the Isolated C-Terminal Domain of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase
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Kim Hak-Jun
Kim Hyun-Woo
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Abstract
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5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly (CTDGG) and gly-ser-ser-gly (CTDGSSG) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), 15N-HSQC, and 31P-NMR revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.
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KEYWORD
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EPSP synthase, herbicide, glyphosate, protein engineering, isothermal titration calorimetry
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