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
摘要：
序列变异和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丝状表型的分子机制。在二氢叶酸还原酶（DHFR）中携带不稳定突变的大肠杆菌菌株。我们发现DHFR活性的部分丧失导致SOS反应，表明DNA损伤和细胞凋亡。这种表型是由脱氧核苷酸水平的不平衡触发的，最突出的是细胞内dTTP的不成比例的下降。我们发现，一个高度合作（希尔系数2.5）的胸苷酸激酶（TMF），下游酶磷酸化dTMP的dTDP的体内活性，是次优dTTP水平的原因。在培养基中补充dTMP挽救了抑制作用，并将体内TMF动力学恢复到几乎完美的Michaelis-Menten，就像其体外动力学一样。总体而言，这项研究突出了细胞环境在塑造酶动力学与细菌表型的系统水平的影响的重要作用。
英文摘要：
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.
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