Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli.
In Escherichia coli, the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) AroG catalyzes the first committed step in the biosynthesis of aromatic compounds. To investigate the feedback inhibition site of AroG, mutated enzymes prepared with sequence-overlap extension PCR were expressed and purified. The enzymatic activity assay showed that the amino acid replacements at Phe144, Leu175, Leu179, Phe209, Trp215Ala and Val221 completely or partially relieved feedback inhibition of AroG addressed by the phenylalanine. Ile10Ala and Delta(1-15) desensitized feedback inhibition and caused a 70 approximately 90% loss of the specific catalytic activities. These results strongly suggest an involvement of the interior region and the N-terminus of the polypeptide chain of AroG in the formation of the feedback inhibition site of DAHPS.
Hu
Changyun
C
Department of Biochemistry, School of Life Sciences, Fudan University, 220 Handan Rd., Shanghai (200433), P. R. China.
Jiang
Peihong
P
Xu
Jianfeng
J
Wu
Yongqing
Y
Huang
Weida
W
D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D025461:Feedback, Physiological; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding; D017384:Sequence Deletion
D004269:DNA, Bacterial; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases