Enzymes: tetrahydrofolate:l-glutamate γ-ligase or folylpolyglutamate synthetase (EC 22.214.171.124); UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-2,6-diaminopimelate ligase; UDP-N-acetylmuramate-l-alanine ligase (EC 126.96.36.199); UDP-N-acetylmuramoyl-l-alanine-d-glutamate ligase (EC 188.8.131.52); UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (EC 184.108.40.206); UDP-N-acetylmuramoyl-l-alanyl-γ-d-glutamyl-meso-2,6-diaminopimelate:d-alanyl-d-alanine ligase (EC 220.127.116.11).
Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments
Article first published online: 20 DEC 2001
European Journal of Biochemistry
Volume 268, Issue 22, pages 5800–5807, November 2001
How to Cite
Dementin, S., Bouhss, A., Auger, G., Parquet, C., Mengin-Lecreulx, D., Dideberg, O., van Heijenoort, J. and Blanot, D. (2001), Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. European Journal of Biochemistry, 268: 5800–5807. doi: 10.1046/j.0014-2956.2001.02524.x
Note: a web site is available at http://www.u-psud.fr/b-430/ibbmc.nsf/
- Issue published online: 20 DEC 2001
- Article first published online: 20 DEC 2001
- (Received 13 June 2001, revised 7 September 2001, accepted 19 September 2001)
- carbamoylated lysine;
- chemical rescue;
- Mur synthetases;
- peptidoglycan biosynthesis
Enzymes MurD, MurE, MurF, folylpolyglutamate synthetase and cyanophycin synthetase, which belong to the Mur synthetase superfamily, possess an invariant lysine residue (K198 in the Escherichia coli MurD numbering). Crystallographic analysis of MurD and MurE has recently shown that this residue is present as a carbamate derivative, a modification presumably essential for Mg2+ binding and acyl phosphate formation. In the present work, the importance of the carbamoylated residue was investigated in MurD, MurE and MurF by site-directed mutagenesis and chemical rescue experiments. Mutant proteins MurD K198A/F, MurE K224A and MurF K202A, which displayed low enzymatic activity, were rescued by incubation with short-chain carboxylic acids, but not amines. The best rescuing agent was acetate for MurD K198A, formate for K198F, and propionate for MurE K224A and MurF K202A. In the last of these, wild-type levels of activity were recovered. A complementarity between the volume of the residue replacing lysine and the length of the carbon chain of the acid was noted. These observations support a functional role for the carbamate in the three Mur synthetases. Experiments aimed at recovering an active enzyme by introducing an acidic residue in place of the invariant lysine residue were also undertaken. Mutant protein MurD K198E was weakly active and was rescued by formate, indicating the necessity of correct positioning of the acidic function with respect to the peptide backbone. Attempts at covalent rescue of mutant protein MurD K198C failed because of its lack of reactivity towards haloacids.