School of Biological Sciences, Victoria University of Wellington, Wellington, Wellington, New Zealand.
Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand.
Elife. 2020 Nov 13;9:e59081. doi: 10.7554/eLife.59081.
Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.
选择一种混杂的酶活性为内源性和新遇到的底物之间的竞争提供了很大的机会，从而影响进化轨迹，这是实验室定向进化研究中经常被忽视的一个方面。我们通过同时随机化八个活性位点残基并检测了约 250,000,000 个重新配置的变体，选择了硝基/醌还原酶 NfsA 来解毒氯霉素。对两种最佳氯霉素还原酶的每一种可能的中间产物的分析揭示了复杂的上位性相互作用。在这两种情况下，只有在 R225 取代后才观察到氯霉素解毒的改善，该取代基本上消除了与内源性醌的活性。易错 PCR 诱变增强了 R225 取代的重要性，在 100%的选择变体中都发现了这种取代。这种强烈的活性权衡表明，内源性细胞代谢物具有很大的潜力来塑造进化结果。未选择的前药转化活性基本不受影响，这强调了负选择对于酶特化的重要性，并为进化基因提供了作为两用可选择/可反选择标记的应用。
