大肠杆菌CMP-唾液酸合成酶最小活性域的研究

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BL21（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－不端的229个氨基酸。在催化活性的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，从而对酶的催化活性与稳定性产生影响。

Minimal functional domain of cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase from Escherichia coli]

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.