Role of the Ortholog and Paralog Amino Acid Invariants in the Active Site of the UDP-MurNAc-L-ALANINE:D-glutamate Ligase (MurD)

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• 作者：Ahmed Bouhss ; Sé ; bastien Dementin ; Claudine Parquet ; Dominique Mengin-Lecreulx ; Jay A. Bertrand ; Dominique Le Beller ; Otto Dideberg ; Jean van Heijenoort ; and Didier Blanot
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：September 21, 1999
• 年：1999
• 卷：38
• 期：38
• 页码：12240 - 12247
• 全文大小：373K
• 年卷期：v.38,no.38(September 21, 1999)
• ISSN：1520-4995

文摘

To evaluate their role in the active site of the UDP-N-acetylmuramoyl-L-alanine:D-glutamateligase (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] orin the sequences of 26 MurD orthologs were submitted to site-directed mutagenesis. All these residueslay 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 theirsteady-state kinetic parameters determined. All had a reduced enzymatic activity, which in many caseswas very low, but no mutation led to a total loss of activity. Examination of the specificity constantsk_cat/K_m for the three MurD substrates indicated that most mutations affected both the binding of onesubstrate and the catalytic process. These kinetic results correlated with the assigned function of the residuesbased on the X-ray structures.