Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli
Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli
复制标题
对酶混杂性的全系统预测揭示了大肠杆菌中吡哆醛 5-磷酸盐生产的新地下替代路线
DOI：
--
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
E. Ruppin
中科院分区：
文献类型：
--
作者：
M. Oberhardt;Raphy Zarecki;L. Reshef;Fangfang Xia;Miquel Duran;Rachel Schreiber;C. Henry;N. Ben;D. Dwyer;U. Gophna;E. Ruppin
最近的见解表明，非特异性和/或混杂的酶在生命中是常见和活跃的。了解这些酶的作用是生物学中一个重要的开放性问题。在这里，我们开发了一个全基因组的方法，PROPER，它使用一个允许的PSI-BLAST方法来预测代谢基因的混杂活动。酶混杂通常使用多拷贝抑制进行实验研究，其中混杂的“混杂”基因的过度表达挽救了由“靶”基因失活引起的致死性。我们使用PROPER预测大肠杆菌中的多拷贝抑制，与已发表的病例（超几何p = 4.4e-13）实现了高度显著的重叠。然后，我们在新的多拷贝抑制实验中验证了三种新的预测靶基因对。接下来，我们超越了PROPER，开发了一种基于网络的方法GEM-PROPER，将PROPER与基因组规模的代谢建模相结合，通过替代代谢途径预测混杂替代。GEM-PROPER预测了一种新的间接酶（thiG），用于生产吡哆醛5 '-磷酸（维生素B6的活性形式）中的必需酶（pdxB），我们通过多拷贝抑制实验验证了这一点。我们对thiG进行了结构分析，以确定其潜在的混杂活性位点，我们通过使相关残基失活并显示出失活活性来实验验证。因此，这项研究是一个成功的例子，其中计算研究导致基于网络的识别关键代谢酶的间接混杂替代，这将是非常难以直接识别的。
Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.
登录
查看更多内容
获得新的代谢能力：大肠杆菌 K-12 中克隆的转氨酶基因的多拷贝抑制。
影响因子：
3.5
作者：
Berg,CM;Wang,MD;Vartak,NB;Liu,L
通讯作者：
Liu,L
影响因子：
2.9
作者：
Settembre, EC;Dorrestein, PC;Ealick, SE
通讯作者：
Ealick, SE
什么是通量平衡分析？
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
10.7
作者：
Patrick, Wayne M.;Quandt, Erik M.;Matsumura, Ichiro
通讯作者：
Matsumura, Ichiro