The evolution of substrate discrimination in macrolide antibiotic resistance enzymes

Abstract

The production of antibiotics by microbes in the environment and their use in medicine andagriculture select for existing and emerging resistance. To address this inevitability, prudentdevelopment 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 enzymeassays, we find that MphI and MphK phosphorylate erythromycin poorly resulting in anantibiotic-sensitive phenotype. Using likelihood reconstruction of ancestral sequences andsite-saturation combinatorial mutagenesis, supported by Mph crystal structures, we determinethat two non-obvious mutations in combination expand the substrate range. Thisapproach should be applicable for studying the functional evolution of any antibiotic resistanceenzyme and for evaluating the evolvability of resistance enzymes to new generations ofantibiotic scaffolds.