Development of the feedback resistant pheA FBR from E. coli and studies on its biochemical characteristics
2016
Cao, T.P., Chosun University School of Medicien, Gwangju, Republic of Korea | Lee, S.H., Korea University, Seoul, Republic of Korea | Hong, K.W., Dongguk University-Seoul, Goyang, Republic of Korea | Lee, S.H., Chosun University School of Medicien, Gwangju, Republic of Korea
The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained pheA FBR gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about 290 μM) for prephenate in comparison with that (about 850 μM) of wild type PheA protein. Kinetic analysis showed that the saturation curve of PheAFBR against phenyalanine is hyperbolic rather than that of PheAWT, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type PheAWT was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.
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