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.
Highlights
The production of antibiotics by microbes in the environment and their use in medicine and agriculture select for existing and emerging resistance
New generations of macrolide antibiotics are being developed to bypass resistance[17,55], which necessitates understanding the evolutionary landscape of Mphs
We find that two non-obvious mutations in tandem are required for expanding the resistance phenotype of MphI to include C3 cladinose macrolides by increasing the catalytic rate
Summary
The production of antibiotics by microbes in the environment and their use in medicine and agriculture select for existing and emerging resistance. 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. For these strategies to be productive in the face of increasing rates of resistance, the functional and evolutionary landscape of resistance enzymes must be interrogated to inform new drug discovery efforts Macrolides such as azithromycin are among the most successful and highly prescribed antibiotics in the world. Mph enzymes inactivate macrolides by phosphorylating the 2′-OH of the essential dimethylamino sugar[27,28], preventing it from binding the ribosome, and providing the chemical rationale for the resistance phenotype
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