Evidence of a functional requirement for a carbamoylated lysine residue inMurD, MurE and MufF synthetases as established by chemical rescue experiments

Enzymes MurD, MurE, MurF, folylpolyglutamate synthetase and cyanophycin syn thetase, which belong to the Mur synthetase superfamily, possess an invaria nt lysine residue (K198 in the Escherichia coli MurD numbering). Crystallog raphic analysis of MurD and MurE has recently shown that this residue is pr esent as a carbamate derivative, a modification presumably essential for Mg 2+ binding and acyl phosphate formation. In the present work, the importanc e of the carbamoylated residue was investigated in MurD, MurE and MurF by s ite-directed mutagenesis and chemical rescue experiments. Mutant proteins M urD K198A/F, MurE K224A and MurF K202A, which displayed low enzymatic activ ity, were rescued by incubation with short-chain carboxylic acids, but not amines. The best rescuing agent was acetate for MurD K198A, formate for K19 8F, and propionate for MurE K224A and MurF K202A. In the last of these, wil d-type levels of activity were recovered. A complementarity between the vol ume of the residue replacing lysine and the length of the carbon chain of t he acid was noted. These observations support a functional role for the car bamate in the three Mur synthetases. Experiments aimed at recovering an act ive enzyme by introducing an acidic residue in place of the invariant lysin e residue were also undertaken. Mutant protein MurD K198E was weakly active and was rescued by formate, indicating the necessity of correct positionin g 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.