Enzyme Efficiency but Not Thermostability Drives Cefotaxime Resistance Evolution in TEM-1 β-Lactamase.
Enzyme Efficiency but Not Thermostability Drives Cefotaxime Resistance Evolution in TEM-1 β-Lactamase.
复制标题
DOI：
10.1093/molbev/msx053
复制
发表时间：
2017-05-01
影响因子：
10.7
通讯作者：
Weinreich DM
中科院分区：
文献类型：
--
作者：
Knies JL;Cai F;Weinreich DM
进化遗传学中一个主要的智力挑战是确定驱动适应的特定表型。酶为探索这个问题提供了一个特别有希望的机会，因为许多酶对生物体健康的贡献取决于相对较少的实验可获得的性质。此外，在第一原理上，对酶的热稳定性的要求与对催化活性的要求是相反的。这一观察结果，再加上酶只有轻微的热稳定性的事实，支持了广泛流行的假设，即能够改善功能的突变需要补偿性突变来恢复热稳定性。在这里，我们第一次明确地验证了这一假设，使用了TEM1β-内酰胺酶中的四个错义突变，这些突变共同将头孢噻肟的最低抑菌浓度(∼)提高了1500倍。首先，我们报告了这些突变的所有组合的酶效率(kcat/Km)和热稳定性(Tm，由此产生的折叠的ΔG)。接下来，我们拟合了一个定量模型，该模型预测了MIC是kCAT/Km和ΔG的函数。虽然Kcat/Km解释了头孢噻肟MIC中∼54%的方差(对数变换后∼为92%)，但ΔG没有提高模型的解释力。我们还发现，头孢噻肟的MIC以kcat/km为单位的上升速度比预期的要慢。对这些差异提出了几种解释。最后，我们在MIC和kcat/Km中展示了实质性的符号上位性，以及表型之间的拮抗多效性，尽管系统中存在近乎数值的可加性。因此，在一个相对“行为良好”的系统中，可以观察到对选择性可达轨迹的限制，以及我们根据潜在机制解释这些限制的能力的限制。
A leading intellectual challenge in evolutionary genetics is to identify the specific phenotypes that drive adaptation. Enzymes offer a particularly promising opportunity to pursue this question, because many enzymes’ contributions to organismal fitness depend on a comparatively small number of experimentally accessible properties. Moreover, on first principles the demands of enzyme thermostability stand in opposition to the demands of catalytic activity. This observation, coupled with the fact that enzymes are only marginally thermostable, motivates the widely held hypothesis that mutations conferring functional improvement require compensatory mutations to restore thermostability. Here, we explicitly test this hypothesis for the first time, using four missense mutations in TEM-1 β-lactamase that jointly increase cefotaxime Minimum Inhibitory Concentration (MIC) ∼1500-fold. First, we report enzymatic efficiency (kcat/KM) and thermostability (Tm, and thence ΔG of folding) for all combinations of these mutations. Next, we fit a quantitative model that predicts MIC as a function of kcat/KM and ΔG. While kcat/KM explains ∼54% of the variance in cefotaxime MIC (∼92% after log transformation), ΔG does not improve explanatory power of the model. We also find that cefotaxime MIC rises more slowly in kcat/KM than predicted. Several explanations for these discrepancies are suggested. Finally, we demonstrate substantial sign epistasis in MIC and kcat/KM, and antagonistic pleiotropy between phenotypes, in spite of near numerical additivity in the system. Thus constraints on selectively accessible trajectories, as well as limitations in our ability to explain such constraints in terms of underlying mechanisms are observed in a comparatively “well-behaved” system.