大肠杆菌CMP-唾液酸合成酶最小活性域的研究

Minimal Functional Domain of Cytidine 5′-monophosphate N-acetylneuraminic Acid (CMP-NeuAc) Synthetase from Escherichia coli

比较大肠杆菌与脑膜炎奈瑟氏球菌的CMP 唾液酸合成酶的氨基酸序列 ,发现大肠杆菌CMP 唾液酸合成酶的保守区域主要位于N 端 ,其C 末端似乎对其催化活性没有作用。通过PCR方法 ,对大肠杆菌CMP 唾液酸合成酶的C 末端进行了一系列截短 ,将得到的产物连接至表达载体pET 15b中 ,在大肠杆菌BL2 1(DE3)pLysS中表达。经IPTG诱导 ,发现从C 末端截去 189个氨基酸酶仍有催化活性 ,说明大肠杆菌CMP 唾液酸合成酶的最小活性域主要集中在N 末端的 2 2 9个氨基酸。有催化活性的C 端缺失突变合成酶的比活、最适pH及热稳定性发生变化 ,提示被截去的C 端氨基酸残基虽不直接参与构成酶的催化活性中心 ,但可影响催化活性域的构象 。

In comparison with its counterpart from N.meningitides, all conserved motifs were found in the N-termini of E.coli CMP-NeuAc synthetase. E.coli CMP-NeuAc synthetase seems to have redundant C-termini with a less effect on its activity. To explain this speculation, a series of recombinant DNAs with deletion from 3'-end of CMP-NeuAc synthetase were produced by PCR, ligated into expression vector pET-15b and expressed in BL21(DE3)pLysS. After induction with IPTG, we found that the recombinant enzyme with deletion of 189 amino acids from C-termini retained its activity. This result demonstrates that the 229 amino acids of N-termini was the minimal functional domain of E.coli CMP-NeuAc synthetase. The deletions altered the optimum pH and thermostability of active truncated enzymes, indicating that the truncated C-terminal amino acids of E.coli CMP-NeuAc synthetase could affect the conformation of the enzymatic catalytic domain and therefore affect its catalytic activity and thermostability, although it is not involved in enzymatic activity directly.