Role of the ortholog and paralog amino acid invariants in the active site of the UDP-MurNAc-L-alanine:D-glutamate ligase (MurD)
| Autor: | Dominique Mengin-Lecreulx, Ahmed Bouhss, Bertrand Ja, Dideberg O, Sébastien Dementin, Claudine Parquet, Le Beller D, van Heijenoort J, Didier Blanot |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Molecular Sequence Data Sequence alignment Biochemistry Conserved sequence Escherichia coli Amino Acid Sequence Binding site Amino Acids Peptide Synthases Peptide sequence Conserved Sequence Alanine chemistry.chemical_classification DNA ligase Binding Sites biology Active site Amino acid Kinetics chemistry Amino Acid Substitution biology.protein Mutagenesis Site-Directed Sequence Alignment |
| Zdroj: | Biochemistry. 38(38) |
| ISSN: | 0006-2960 6223-6229 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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