Understanding the evolutionary optimization of enzyme dynamics and specificity

相比之下，蛋白质设计界历来以高度稳定和僵硬的蛋白质支架为目标。虽然该领域已经实现了以原子级别的精度设计蛋白质的能力，但事实证明，创造生物和工业相关水平的酶功能更加难以捉摸。CDM13是一种简单、动态的自组装“预先进化”的双核金属蛋白，能够执行许多差异很大的催化反应。这种混杂的原始酶提供了一个独特的模型系统来详细研究早期的酶是什么样子，并检查功能序列空间中连接这些原始酶和现在的酶的途径。该项目不仅将增进对天然双核酶进化和功能的了解，而且还将产生一系列强大的双核酶，这些酶可能在未来的微生物生物燃料项目中被证明是有用的，并作为环境友好的羟基化催化剂。作为该项目的一部分，本科生、研究生和纽约市公立高中教师将在一个机构接受生物物理学跨学科科学方面的培训，该机构的学生人数由55%未被充分代表的少数民族组成。CDM13是一种简单、动态的自组装“进化前”双核金属蛋白，能够在溶液和体内执行许多截然不同的催化任务，其速度接近于在催化这些反应的天然双核酶中观察到的速度，包括作为双铁酶的甲烷羟化和芳胺氧化以及作为双锰酶的过氧化氢氧化。这类蛋白质是理想的，因为天然双核酶具有几乎可叠加的主干结构，因此进化出的催化功能的某些方面可以很容易地移植。这个项目的中心是使用CDM13来观察在单一支架上几种不同形式的多电子催化上质子的特异性、动力学和生物能量耦合的演变，积极地识别酶功能和特异性所需的关键特征，并为我们提供一个新的窗口，了解现代酶是如何进化其底物和结构细节的。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Enzymes, the protein catalysts for biological reactions, are able to efficiently perform difficult chemical transformations with a high degree of specificity. The evolution of enzymatic function is believed to begin with a dynamic promiscuous enzyme, which then evolves to be more rigid and specific. By contrast, the protein design community has historically targeted highly stable and inflexible protein scaffolds. While the field has achieved the ability to design proteins with atomic-level precision, the creation of biologically and industrially relevant levels of enzymatic function has proven more elusive. CDM13 is simple, dynamic self-assembling ‘pre-evolved’ binuclear metalloprotein capable of performing a number of widely differing catalytic reactions. This promiscuous primordial enzyme provides a unique model system to study in detail what early enzymes were like and to examine the pathways in functional sequence space that connects these primordial enzymes to present day enzymes. This project will not only generate an improved understanding of natural binuclear enzyme evolution and function, but also in a palette of robust binuclear enzymes, which may prove useful in future microbial biofuel projects and as environmentally friendly hydroxylation catalysts. As part of the project undergraduates, graduate students and New York City Public High School teachers will be trained in the interdisciplinary science of biophysics at an institution with a student body which is composed of 55% underrepresented minorities. An outreach plan is presented which includes the development of a new planetarium demonstration of protein structure and dynamics. CDM13 is a simple, dynamic self-assembling ‘pre-evolved’ binuclear metalloprotein capable of performing a number of widely differing catalytic tasks in solution and in vivo at rates that approach those observed in the natural binuclear enzymes that catalyze these reactions, including methane hydroxylation and arylamine oxidation as a diiron enzyme and hydrogen peroxide oxidation as a di-manganese enzyme. The binuclear enzyme family members each have two metal ions bound within a D2-symmetric antiparallel four alpha helical bundle. This class of proteins is ideal for because the natural binuclear enzymes have almost superimposable backbone structures, and thus aspects of the evolved catalytic functions can be easily transplanted. This project centers on the use of CDM13 to look at the evolution of specificity, dynamics, and bioenergetic coupling to protons on several different forms of multielectron catalysis in a single scaffold, positively identifying critical features necessary for enzyme function and specificity and giving us a new window on how modern enzymes have evolved their substrate and structural specificity.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.