论文标题
对点突变的代谢反应揭示了体内酶活性和表型调节的原理
Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype
论文作者
论文摘要
序列变异与表型之间的关系知之甚少。在这里，我们使用代谢组学分析来阐明大肠杆菌菌株的丝状表型的分子机制，这些丝状菌株携带二氢叶酸还原酶（DHFR）中携带不稳定的突变。我们发现DHFR活性的部分丧失会导致SOS响应指示DNA损伤和细胞丝。这种表型是由脱氧核苷酸水平的不平衡引起的，最显着的细胞内DTTP下降。我们表明，高度合作（山系数2.5）在胸苷酸酯激酶（TMK）的体内活性（TMK）是一种下游酶，将DTMP磷酸化为DTDP，是次优DTTP水平的原因。媒体中的DTMP补充可挽救细丝，并在体内TMK动力学中恢复几乎完美的Michaelis-Menten，就像其体外动力学一样。总体而言，这项研究强调了细胞环境在雕刻具有系统水平对细菌表型的影响的酶动力学中的重要作用。
The relationship between sequence variation and phenotype is poorly understood. Here we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in the Dihydrofolate Reductase (DHFR). We find that partial loss of DHFR activity causes SOS response indicative of DNA damage and cell filamentation. This phenotype is triggered by an imbalance in deoxy nucleotide levels, most prominently a disproportionate drop in the intracellular dTTP. We show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Thymidylate Kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP, is the cause of suboptimal dTTP levels. dTMP supplementation in the media rescues filamentation and restores in vivo Tmk kinetics to almost perfect Michaelis-Menten, like its kinetics in vitro. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system level implications for bacterial phenotype.
