High-Order Epistasis in Catalytic Power of Dihydrofolate Reductase Gives Rise to a Rugged Fitness Landscape in the Presence of Trimethoprim Selection
High-Order Epistasis in Catalytic Power of Dihydrofolate Reductase Gives Rise to a Rugged Fitness Landscape in the Presence of Trimethoprim Selection
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DOI：
10.1093/molbev/msz086
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发表时间：
2019-07-01
影响因子：
10.7
通讯作者：
Toprak，Erdal
中科院分区：
文献类型：
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作者：
Tamer，Yusuf Talha;Gaszek，Ilona K.;Toprak，Erdal
几种抗生素靶蛋白的进化适应图已经被全面绘制，显示了突变之间的强烈高位上位性，但在生化和结构水平上对这些影响的理解仍然是开放的。在这里，我们开展了一项广泛的实验和计算研究，以定量地了解在甲氧苄氨嘧啶诱导的选择存在的情况下，大肠埃希氏菌的DHFR酶的进化动力学。为了促进这一点，我们开发了一种新的体外快速表征DHFR稳态动力学的方法。针对横跨DHFR底物结合口袋的总共10个残基的抗性突变的生化和结构表征表明，突变的DHFR酶的催化效率发生了明显的变化。接下来，我们测量了突变体库的生化参数(Km、KI和KCAT)，该库携带了六个与DHFR抗药性相关的突变的所有可能组合，并量化了它们之间的上位性相互作用。我们发现DHFR(KcatandKm)催化能力的高阶上位性在甲氧苄啶选择下创造了一个崎岖的适应度图景。综上所述，我们的数据提供了一个具体的例子，说明了生化参数水平的上位性耦合如何产生复杂的适应度格局，并为开发突变特异性抑制剂提出了新的策略。
Evolutionary fitness landscapes of several antibiotic target proteins have been comprehensively mapped showing strong high-order epistasis between mutations, but understanding these effects at the biochemical and structural levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics ofEscherichia colidihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim-induced selection. To facilitate this, we developed a new in vitro assay for rapidly characterizing DHFR steady-state kinetics. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct changes in the catalytic efficiencies of mutated DHFR enzymes. Next, we measured biochemical parameters (Km,Ki, andkcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the high-order epistasis in catalytic power of DHFR (kcatandKm) creates a rugged fitness landscape under trimethoprim selection. Taken together, our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors.