Structure-based site-directed mutagenesis of the UDP-MurNAc-pentapeptide-binding cavity of the FemX alanyl transferase from Weissella viridescens.
Weissella viridescens FemX (FemX(Wv)) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX(Wv) in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX(Wv) transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal D-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal D-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.
Maillard
Antoine P
AP
Laboratoire de Recherche Moléculaire sur les Antibiotiques, INSERM U655, Université Paris 6, Paris, France.
Biarrotte-Sorin
Sabrina
S
Villet
Régis
R
Mesnage
Stéphane
S
Bouhss
Ahmed
A
Sougakoff
Wladimir
W
Mayer
Claudine
C
Arthur
Michel
M
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D003461:Crystallography; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019884:Nitrogenous Group Transferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*
D014538:Uridine Diphosphate N-Acetylmuramic Acid; C002225:UDP-N-acetylmuramic acid pentapeptide; D019884:Nitrogenous Group Transferases; C476453:UDP-N-acetylmuramoyl pentapeptide-lysine N(6)-alanyltransferase