Investigation of the ATP Binding Site ofEscherichia coliAminoimidazole Ribonucleotide Synthetase Using Affinity Labeling and Site-Directed Mutagenesis†
ABSTRACT
Macrolide 2′-phosphotransferase [MPH(2′)] transfers the γ phosphate of ATP to the 2′-OH group of macrolide antibiotics. The role of aspartic acids in the putative ATP-binding site of MPH(2′)II was investigated through the substitution of alanine for aspartate by site-directed mutagenesis. D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme.
Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II