Effect of F209S Mutation of Escherichia coli AroG on Resistance to Phenylalanine Feedback Inhibition.
In Escherichia coli, 80% of the 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase was encoded by aroG gene. The aroG gene was amplified by polymerase chain reaction(PCR) from strain K-12 and a mutant strain resistant to phenylalanine analogues. The PCR products were cloned and subject to DNA sequence analysis. A single base mutation of Tright curved arrow C was detected at nucleotide 625, which causes a substitution of Phe(209) by Ser in the gene product. The gene was expressed on pTrc99A in E.coli strain JM105. Under the induction of IPTG, distinct band with the expected molecule weight was detected on SDS-polyacrylamide gel electrophoresis and the specific activity of DAHP of the crude extract of the transformed cells increased by 1.8-fold. Enzyme activity inhibition analysis revealed the high resistance of mutant AroG to feedback inhibition by phenylalanine. JM105 cells harboring with mutant aroG gene showed were able to grow on medium containing higher concentration of analogues than that carrying normal aroG gene. Discussion was focused on the varieties of mutations contributing to desensitization of feedback inhibition.
Jiang
Pei-Hong
PH
Department of Biochemistry, School of Life Science, Fudan University, Shanghai 200433, China. whuang@fudan.edu.cn
Shi
Mi
M
Qian
Zhi-Kang
ZK
Li
Ning-Jun
NJ
Huang
Wei-Da
WD