Hu Changyun, Jiang Peihong, Xu Jianfeng, Wu Yongqing, Huang Weida
Department of Biochemistry, School of Life Sciences, Fudan University, 220 Handan Rd., Shanghai (200433), P. R. China.
J Basic Microbiol. 2003;43(5):399-406. doi: 10.1002/jobm.200310244.
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.
在大肠杆菌中，苯丙氨酸敏感的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶（DAHPS）AroG催化芳香族化合物生物合成中的首个关键步骤。为了研究AroG的反馈抑制位点，利用序列重叠延伸PCR制备的突变酶进行了表达和纯化。酶活性测定表明，Phe144、Leu175、Leu179、Phe209、Trp215Ala和Val221处的氨基酸替换完全或部分解除了苯丙氨酸对AroG的反馈抑制。Ile10Ala和Delta(1-15)使反馈抑制脱敏，并导致比催化活性损失约70%至90%。这些结果有力地表明，AroG多肽链的内部区域和N末端参与了DAHPS反馈抑制位点的形成。
