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-alanine:D-glutamate ligase (MurD) from Escherichia coli, 12 residues conserved either in the Mur superfamily [Eveland, S. S., Pompliano, D. L., and Anderson, 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 site-directed mutagenesis. All these residues lay within the cleft of the active 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, E157A/K, H183A, Y194F, K198A/F, N268A, N271A, H301A, R302A, D317A, and R425A) containing a C-terminal (His)(6) extension were prepared and their steady-state kinetic parameters determined. All had a reduced enzymatic activity, which in many cases was very low, but no mutation led to a total loss of activity. Examination of the specificity constants k(cat)/K(m) for the three MurD substrates indicated that most mutations affected both the binding of one substrate and the catalytic process. These kinetic results correlated with the assigned function of the residues based on the X-ray structures.
Bouhss
A
A
EP 1088 du CNRS, Biochimie Structurale et Cellulaire, Université de Paris-Sud, Orsay, France.
Dementin
S
S
Parquet
C
C
Mengin-Lecreulx
D
D
Bertrand
J A
JA
Le Beller
D
D
Dideberg
O
O
van Heijenoort
J
J
Blanot
D
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics*; D016415:Sequence Alignment
D000596:Amino Acids; D010453:Peptide Synthases; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase