A Single Molecule Perspective on the Functional Diversity of in Vitro Evolved β-Glucuronidase
A Single Molecule Perspective on the Functional Diversity of in Vitro Evolved β-Glucuronidase
复制标题
DOI：
10.1021/ja412379p
复制
发表时间：
2014-04-23
影响因子：
15
通讯作者：
Gorris, Hans H.
中科院分区：
文献类型：
--
作者：
Liebherr, Raphaela B.;Renner, Max;Gorris, Hans H.
通过在单分子水平上比较野生型和体外进化的β-葡萄糖醛酸苷酶（GUS）的动力学，研究了驱动新酶活性进化的机制。数百个单一的GUS分子被分离在62 500个超小反应室蚀刻到熔融石英载玻片的表面的大阵列，以观察他们的个人基板周转率在平行的荧光显微镜。单个GUS分子具有长寿命但不同的活性状态，并且它们的平均活性与经典的Michaelis-Menten动力学一致。大量的单分子底物周转率代表了整个酶群体内的活性分布。部分进化的GUS在单个酶分子中显示出比野生型GUS宽得多的活性分布。更广泛的活动分布表明，在一个部分进化的酶，作为所谓的generalists的特点是其混杂的活动与许多不同的底物群体中的单个分子之间的工作的功能分工。
The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved beta-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62 500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.