Related Experiment Videos
Identification of functional amino acids in the macrolide 2'-phosphotransferase II
K Taniguchi1, A Nakamura, K Tsurubuchi
1Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan.
Abstract:
Macrolide 2'-phosphotransferase [MPH(2')] transfers the gamma 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.
Insights
Investigating aspartic acids in macrolide 2'-phosphotransferase (MPH(2')) revealed their crucial role in enzyme activity. Mutations at key aspartate sites abolished oleandomycin inactivation, highlighting their importance in antibiotic resistance mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Macrolide 2 phosphotransferase (MPH(2 )) confers resistance to macrolide antibiotics by phosphorylating them.
- The enzyme's catalytic mechanism and substrate binding, particularly the role of the ATP-binding site, remain incompletely understood.
Purpose of the Study:
- To elucidate the function of aspartic acid residues within the putative ATP-binding site of MPH(2 )II.
- To determine the impact of specific aspartate mutations on the enzymatic activity of MPH(2 )II.
Main Methods:
- Site-directed mutagenesis was employed to substitute conserved aspartate residues with alanine.
- Enzymatic activity assays were performed using oleandomycin as the substrate to assess the function of mutant MPH(2 )II enzymes.
Main Results:
- Mutant strains D200A, D209A, D219A, and D231A exhibited complete loss of MPH(2 )II activity against oleandomycin.
- The D227A mutant retained approximately 7% of the wild-type enzyme's phosphotransferase activity.
Conclusions:
- Aspartic acid residues at positions 200, 209, 219, and 231 are essential for the catalytic function of MPH(2 )II.
- Aspartic acid at position 227 plays a significant, but not entirely indispensable, role in MPH(2 )II activity, suggesting a complex active site structure.