Minimal functional domain of cytidine 5''-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase from Escherichia coli]
大肠杆菌CMP-唾液酸合成酶最小活性域的研究

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.