Castro-Fernandez Víctor, Herrera-Morande Alejandra, Zamora Ricardo, Merino Felipe, Gonzalez-Ordenes Felipe, Padilla-Salinas Felipe, Pereira Humberto M, Brandão-Neto Jose, Garratt Richard C, Guixe Victoria
From the Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 800003, Chile,
From the Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 800003, Chile.
J Biol Chem. 2017 Sep 22;292(38):15598-15610. doi: 10.1074/jbc.M117.790865. Epub 2017 Jul 18.
One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders , , and , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in .
分子进化的一个核心目标是确定导致酶改变其底物特异性的机制和进化力量；然而，这些过程在很大程度上仍未得到探索。以古菌的糖酵解ADP依赖性激酶为模型，包括 、 和 目，并采用古酶学、进化统计学和蛋白质结构分析相结合的方法，我们能够追踪ADP依赖性激酶进化过程中底物特异性的变化以及这些变化的结构决定因素。为此，我们研究了五种关键的复活祖先酶及其现存对应物。我们发现，从一个可以将葡萄糖或6-磷酸果糖（果糖-6-P）作为底物进行磷酸化的双功能祖先到一个6-磷酸果糖特异性酶的功能重大转变，是由单个氨基酸取代引发的，该取代导致对葡萄糖的基态模式下的负选择，以及祖先蛋白特异性随后1600倍的变化。这种变化使葡萄糖的残留磷酸化成为一种混杂且生理上无关紧要的活性，突出了混杂性可能是祖先酶活性的进化遗迹，随着时间的推移已被消除。我们还可以通过使用离散二元特征的进化模型重建底物利用的进化历史，这表明在酶进化过程中底物利用可以离散地丢失或获得。这些发现例证了负选择和细微的酶变化如何导致功能上的重大进化转变，随后可以产生重要的适应性优势，例如，在提高 中的糖酵解效率方面。
