Role of the ortholog and paralog amino acid invariants in the active site of the UDP-MurNAc-L-alanine : D-glutamate ligase (MurD)

To evaluate their role in the active site of the UDP-N-acetylmuramoyl-L-ala nine:D-glutamate ligase (MurD) from Escherichia coli, 12 residues conserved either in the Mur superfamily [Eveland, S. S.. Pompliano, D. L., and Ander son, M. S. (1997) Biochemistry 36, 6223-6229; Bouhss, A., Mengin-Lecreulx, D., Blanot, D., van Heijenoort, J., and Parquet, C. (1997) Biochemistry 36, 11556-11563] or in the sequences of 26 MurD orthologs were submitted to si re-directed mutagenesis. All these residues lay within the cleft of the act ive site of MurD as defined by its 3D structure [Bertrand, J. A., Auger, D. , Fanchon, E., Martin, L., Blanot, D., van Heijenoort, J., and Dideberg, O. (1997) EMBO J. 16, 3416-3425]. Fourteen mutant proteins (D35A, K115A, E157 A/K, H183A, Y194F, K198A/F, N268A, N271A, H301A, R302A, D317A, and R425A) c ontaining a C-terminal (His)(6) extension were prepared and their steady-st are kinetic parameters determined. All had a reduced enzymatic activity, wh ich in many cases was very low, but no mutation led to a total loss of acti vity. Examination of the specificity constants k(cat)/k(m) for the three Mu rD substrates indicated that most mutations affected both the binding of on e substrate and the catalytic process. These kinetic results correlated wit h the assigned function of the residues based on the X-ray structures.