The Rosetta Enzyme ReDesign: Targeting PON1 as OP Scavenger

Rosetta 酶重新设计：将 PON1 作为 OP 清除剂

• 批准号: 7870162
• 负责人: Jennifer M Bui
• 金额: $ 8.41万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7870162
• 关键词: Active Sites; Agriculture; Algorithms; Amino Acids; Biochemistry; Bioinformatics; Biological; Biotechnology; Catalysis; Cessation of life; Chemicals; Cholinesterase Inhibitors; Collaborations; Development; Ecosystem; Engineering; Ensure; Environmental Health; Environmental Protection; Enzymes; Equilibrium; Evolution; Exposure to; Fellowship; Flame Retardants; Gasoline; Generations; Genetic Polymorphism; Gluconolactonase; Goals; Health; Human; Human Engineering; Hydrolysis; Individual; Insecticides; Institution; Interdisciplinary Study; Laboratories; Letters; Link; Methodology; Methods; Mothers; Nature; Organophosphates; Performance; Pesticides; Phosphoric Triester Hydrolases; Physiological; Plastics; Population; Positioning Attribute; Process; Property; Protein Dynamics; Protein Engineering; Proteins; Reaction; Research; Research Project Grants; Research Proposals; Role; Rubber; Serum; Site; Specificity; Substrate Specificity; Temperature; Testing; Therapeutic; Toxic effect; Training; aryldialkylphosphatase; bioscavenger; career; conformer; design; esterase; novel strategies; preference; professor; public health relevance; research study; restraint; stem; structural biology; theories; working group

DESCRIPTION (provided by applicant) Many organophosphate (OP) compounds are potent cholinesterase inhibitors and have extensively been used as pesticides and insecticides in agriculture, as a flame retardant in plastics and rubbers, and even as a gasoline additive [1-3]. Each year, there are approximately three million pesticide poisonings world-wide resulting in more than 200,000 deaths [1]. Long term exposures to these OP compounds in commercial products have been linked to several adverse health effects and permanent damages to our ecosystem. The search for enzymes that act as bioscavengers against toxic OP compounds has become an intense research topic. In this context, human serum paraoxonase, PON1 could become a promising scavenger against highly toxic OP compounds because 1) it is in human serum, 2) it can hydrolyze OP compounds, and 3) it displays large pH optima and performs optimally at physiological temperature [5-7]. However, PON1's physiological role has not yet been unambiguously identified. It is considered to be a promiscuous enzyme [6]. In addition, PON1 is polymorphic in human populations and different individuals also express widely different levels of this enzyme [5]. The proposed research is to re-engineer PON1 using a new approach that integrates both conformational dynamics and evolution information by 1) characterizing its catalytic activities, 2) identifying conformational diversities of the enzyme, 3) exploring the effects of polymorphisms on substrate specificity and stability, and 4) integrating these enzymatic properties into the Rosetta Enzyme Design method [10, 11]. The proposed research will therefore, not only focus on characterizing reaction mechanisms of PON1 at its active site, but also include dynamical information beyond this site, such that the redesigned enzyme will be more catalytic efficient and highly expressible. The resulting computational design will concurrently be tested and characterized in the wet-lab. The experimental biochemistry will be included as restraint information to optimize the designed cycle further. The research proposal is the interplay between theory, experiments, and computations which will facilitate a rapid progress for the proposed research. The candidate's main career goals are to become a tenured, endowed professor at a Tier 1 research institution and to have a multidisciplinary research group that works on methodology developments and applications to environmental health sciences and biotechnology. She believe that in less than a decade, computational structural biology and cellular bioinformatics will have come together to advance research in protein engineering; at this point, in her research plans for the K01 fellowship, she aim to get the right breadth of expertise to be positioned precisely at that intersection. In this context, she is eager to take her computational expertise developed in the McCammon and Dobson laboratories and apply it to the enzyme design that Professor Baker, one of the world renowned expert in the field, is pursuing. Public Health Relevance: The new Rosetta Enzyme Design is introduced and applied to re-engineer the human paraoxonase to be used as effective bioscavengers that sequester highly toxic organophosphate compounds commonly found in commercial products. This proposal thus has direct relevance impact in environmental protections and biotechnologies.

描述（由申请人提供） 许多有机磷酸酯（OP）化合物是有效的胆碱酯酶抑制剂，并且已广泛用作农业中的杀虫剂和杀虫剂，用作塑料和橡胶中的阻燃剂，甚至用作汽油添加剂[1-3]。每年，全世界约有300万起农药中毒事件，导致20多万人死亡[1]。长期暴露于商业产品中的这些OP化合物与几种不良健康影响和对我们生态系统的永久性损害有关。寻找作为生物清除剂对抗有毒OP化合物的酶已成为一个激烈的研究课题。 在这种情况下，人血清对氧磷酶，PON 1可能成为一种有前途的清除剂，可以清除高毒性OP化合物，因为1）它存在于人血清中，2）它可以水解OP化合物，3）它显示出大的最佳pH值，并在生理温度下表现最佳[5-7]。 然而，PON 1的生理作用尚未明确确定。 它被认为是一种混杂的酶[6]。 此外，PON 1在人群中具有多态性，不同个体也表达广泛不同水平的这种酶[5]。拟议的研究是使用一种新方法重新设计PON 1，该方法通过1）表征其催化活性，2）鉴定酶的构象差异，3）探索多态性对底物特异性和稳定性的影响，以及4）将这些酶特性整合到Rosetta酶设计方法中[10，11]。因此，拟议的研究不仅关注PON 1在其活性位点的反应机制，而且还包括该位点以外的动力学信息，这样重新设计的酶将更具催化效率和高表达性。由此产生的计算设计将同时在湿实验室进行测试和表征。实验生物化学将被包括作为约束信息，以进一步优化设计的循环。研究建议是理论，实验和计算之间的相互作用，这将有助于拟议的研究的快速进展。 候选人的主要职业目标是成为一级研究机构的终身教授，并拥有一个多学科研究小组，致力于环境健康科学和生物技术的方法开发和应用。 她认为，在不到十年的时间里，计算结构生物学和细胞生物信息学将共同推进蛋白质工程的研究;在这一点上，在她的K 01奖学金的研究计划中，她的目标是获得正确的专业知识广度，以准确定位在该交叉点。在这种情况下，她渴望把她在McCammon和多布森实验室开发的计算专业知识应用于该领域的世界知名专家之一Baker教授正在追求的酶设计。 公共卫生相关性：新的Rosetta酶设计被引入并应用于重新设计人类对氧磷酶，以用作有效的生物清除剂，隔离商业产品中常见的高毒性有机磷酸盐化合物。因此，该提案在环境保护和生物技术方面具有直接的相关影响。