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Updated: Feb 15, 2026
Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
The evolution of substrate discrimination in macrolide antibiotic resistance enzymes
Andrew C Pawlowski1, Peter J Stogios2,3, Kalinka Koteva1
1Michael G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, L8S 4L8, ON, Canada.
Abstract:
The production of antibiotics by microbes in the environment and their use in medicine and agriculture select for existing and emerging resistance. To address this inevitability, prudent development of antibiotic drugs requires careful consideration of resistance evolution. Here, we identify the molecular basis for expanded substrate specificity in MphI, a macrolide kinase (Mph) that does not confer resistance to erythromycin, in contrast to other known Mphs. Using a combination of phylogenetics, drug-resistance phenotypes, and in vitro enzyme assays, we find that MphI and MphK phosphorylate erythromycin poorly resulting in an antibiotic-sensitive phenotype. Using likelihood reconstruction of ancestral sequences and site-saturation combinatorial mutagenesis, supported by Mph crystal structures, we determine that two non-obvious mutations in combination expand the substrate range. This approach should be applicable for studying the functional evolution of any antibiotic resistance enzyme and for evaluating the evolvability of resistance enzymes to new generations of antibiotic scaffolds.
Insights
Understanding antibiotic resistance is crucial. This study reveals how two mutations in macrolide kinase MphI expand its substrate range, offering insights into resistance evolution and new drug development.
Area of Science:
- Microbiology
- Biochemistry
- Evolutionary Biology
Background:
- Antibiotic resistance is a growing threat driven by microbial production and clinical/agricultural use of antibiotics.
- Understanding the evolution of antibiotic resistance mechanisms is vital for developing new drugs.
Purpose of the Study:
- To identify the molecular basis for expanded substrate specificity in the macrolide kinase MphI.
- To investigate the evolutionary pathways leading to altered antibiotic resistance enzyme function.
Main Methods:
- Phylogenetic analysis
- Drug-resistance phenotype determination
- In vitro enzyme assays
- Ancestral sequence reconstruction
- Site-saturation mutagenesis
- Analysis of Mph crystal structures
Main Results:
- MphI and MphK poorly phosphorylate erythromycin, resulting in an antibiotic-sensitive phenotype.
- Two specific mutations were identified as key to expanding MphI's substrate range.
- This provides a molecular explanation for altered macrolide kinase activity.
Conclusions:
- The study elucidates the evolutionary mechanism behind expanded substrate specificity in antibiotic resistance enzymes.
- The findings offer a framework for studying the evolution of resistance and designing next-generation antibiotics.
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