Pawlowski Andrew C, Stogios Peter J, Koteva Kalinka, Skarina Tatiana, Evdokimova Elena, Savchenko Alexei, Wright Gerard D
Michael G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, L8S 4L8, ON, Canada.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, M5S 3E5, ON, Canada.
Nat Commun. 2018 Jan 9;9(1):112. doi: 10.1038/s41467-017-02680-0.
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.
环境中微生物产生抗生素以及它们在医学和农业中的使用会选择现有的和新出现的耐药性。为应对这种不可避免的情况，谨慎开发抗生素药物需要仔细考虑耐药性的演变。在这里，我们确定了MphI中底物特异性扩展的分子基础，MphI是一种大环内酯激酶（Mph），与其他已知的Mph不同，它不会赋予对红霉素的耐药性。通过系统发育学、耐药表型和体外酶分析相结合的方法，我们发现MphI和MphK对红霉素的磷酸化能力较差，导致抗生素敏感表型。利用祖先序列的似然重建和位点饱和组合诱变，并结合Mph晶体结构，我们确定两个不明显的突变共同作用扩大了底物范围。这种方法应该适用于研究任何抗生素耐药酶的功能进化，以及评估耐药酶对新一代抗生素支架的进化能力。
