Comprehensive exploration of the sequence-structure-function relationships within the nitroreductase superfamily.

全面探索硝基还原酶超家族内的序列-结构-功能关系。

• 批准号: RGPIN-2017-04909
• 负责人: Tokuriki, Nobuhiko
• 金额: $ 3.64万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=666932
• 关键词: Comprehensive; exploration; sequence; structure; function

Background: The remarkable ability of enzymes to effectively catalyze diverse chemical reactions is one of the critical foundations of biology; more than that, it offers an abundance of biotechnological applications. At this point, however, we understand only “the tip of the iceberg” in terms of the vast functional diversity found in nature, and as such the exploration of uncharacterized enzymes is extremely important. The nitroreductase (NTR) superfamily is a historically neglected, but biologically and biotechnologically important, group of evolutionarily related enzymes. Despite the biological importance of these enzymes, historical research efforts have focused on only a few functional families, while the vast majority of enzymes remain uncharacterized.******Research Aims: In this research program, I aim to substantially build our knowledge of functional diversity and sequence-structure-function relationships of NTR enzymes. To this end, I have established two state-of-the-art large-scale enzyme characterization approaches that will provide both “global” and “high-resolution” insights. The integration of these two complementary approaches will reveal the “molecular blueprints” for NTR enzymes, and improve our ability to predict, design and engineer synthetic proteins that accomplish the functions of NTR enzymes.***Aim 1. Perform large-scale profiling of enzyme activity for more than 600 enzymesacross 22 NTR subgroups, as well as bioinformatically reconstructed ancestral NTR sequences. We will reveal the “global” sequence-structure-function relationships of enzymes in the NTR superfamily, and identify the key molecular signatures that result in functional transitions. In addition, we will identify, verify and discover NTR functions, including those from as yet uncharacterized families. ***Aim 2. Conduct extraordinarily deep mutational analysis on representative NTR enzymes. We will generate mutational libraries of key NTR enzymes, and characterize the functional effect of each mutation using high-throughput screening and sequencing technologies. This will enable us to analyze “high-resolution” molecular architecture and will reveal the residues and mutations responsible for evolutionary functional transitions.******Impacts: Our proposed program will have impacts that are both significant and broad: First, we will generate invaluable information that advances our knowledge of the biological and chemical functions and evolutionary divergence of NTR enzymes. Second, we will develop an “NTR toolbox” which will have applications in diverse industrial and biomedical settings. Finally, our novel and innovative approach characterizing superfamily-wide functions will be a milestone in the field and aid those seeking similar levels of comprehensive understanding for other protein superfamilies.

背景：酶能有效催化多种化学反应是生物学的重要基础之一；不仅如此，它还提供了丰富的生物技术应用。然而，在这一点上，就自然界中发现的巨大功能多样性而言，我们只了解“冰山一角”，因此探索未表征的酶是极其重要的。硝基还原酶（NTR）超家族是历史上被忽视的，但在生物学和生物技术上是重要的，一组进化相关的酶。尽管这些酶具有重要的生物学意义，但历史上的研究工作只集中在几个功能家族上，而绝大多数酶仍未被表征。******研究目标：在这个研究项目中，我的目标是实质性地建立我们对NTR酶的功能多样性和序列-结构-功能关系的认识。为此，我建立了两种最先进的大规模酶表征方法，将提供“全局”和“高分辨率”的见解。这两种互补方法的整合将揭示NTR酶的“分子蓝图”，并提高我们预测、设计和工程合成蛋白质的能力，以完成NTR酶的功能。* * *的目标1。对22个NTR亚群中超过600种酶的酶活性进行大规模分析，以及生物信息学上重建祖先NTR序列。我们将揭示NTR超家族中酶的“全局”序列-结构-功能关系，并确定导致功能转变的关键分子特征。此外，我们将识别，验证和发现NTR功能，包括那些尚未表征的家族。* * *的目标2。对代表性的NTR酶进行异常深入的突变分析。我们将建立关键NTR酶的突变文库，并利用高通量筛选和测序技术表征每个突变的功能效应。这将使我们能够分析“高分辨率”的分子结构，并将揭示负责进化功能转变的残基和突变。******影响：我们提出的项目将产生重大而广泛的影响：首先，我们将产生宝贵的信息，促进我们对NTR酶的生物和化学功能以及进化差异的了解。其次，我们将开发一个“NTR工具箱”，将在各种工业和生物医学环境中得到应用。最后，我们的新颖和创新的方法表征全超家族功能将是该领域的一个里程碑，并帮助那些寻求对其他蛋白质超家族的类似水平的全面理解。