Minimal functional domain of cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase from Escherichia coli.
- Author:
Chun-Sheng JIN
1
;
Cheng JIN
Author Information
1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Cytidine Monophosphate N-Acetylneuraminic Acid;
metabolism;
Enzyme Stability;
Escherichia coli;
enzymology;
Hydrogen-Ion Concentration;
Molecular Sequence Data;
Mutation;
N-Acylneuraminate Cytidylyltransferase;
chemistry;
physiology;
Structure-Activity Relationship
- From:
Chinese Journal of Biotechnology
2002;18(6):676-682
- CountryChina
- Language:Chinese
-
Abstract:
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 C0termini 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.
