摘要

微生物在环境中生产抗生素及其在医药和农业中的应用，可以选择现有的和正在形成的抗药性。为了解决这种必然性，谨慎开发抗生素药物需要仔细考虑抗药性的演变。在这里，我们确定扩大底物特异性的分子基础，MphI是一种大环内酯类激酶（Mph），与其他已知Mphs相比，它不赋予对红霉素的抗性。结合系统发育，药物抗性表型和体外酶测定，我们发现MphI和MphK磷酸化红霉素效果不佳，导致抗生素敏感表型。利用Mph晶体结构支持的祖先序列的可能性重构和位点饱和度组合诱变，我们确定两种非显而易见的突变组合扩大了底物范围。这种方法应该适用于研究任何抗生素抗性酶的功能进化和评估新一代抗生素支架抗性酶的可进化性。

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.


https://www.nature.com/articles/s41467-017-02680-0