The Rosetta Enzyme ReDesign: Targeting PON1 as OP Scavenger

Rosetta 酶重新设计：将 PON1 作为 OP 清除剂

基本信息

批准号：
8573990
负责人：
Jennifer M Bui
金额：
$ 8.36万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8573990
关键词：
Active Sites Agriculture Algorithms Amino Acids Biochemistry Bioinformatics 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 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

项目摘要

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）许多有机磷酸盐（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农药和杀虫剂，用作塑料和橡胶的阻燃剂，甚至用作汽油添加剂[1-3]。每年，全球大约有300万种农药中毒导致200,000多人死亡[1]。这些商业产品中这些OP化合物的长期暴露与我们的生态系统的几种不利健康影响和永久性损害有关。寻找作为对有毒OP化合物的生物库助剂的酶已成为一个激烈的研究主题。在这种情况下，人血清二氧酮酶PON1可能成为对剧毒OP化合物的有前途的清道夫，因为1）它在人血清中，2）它可以水解OP化合物，3）它显示出较大的pH值，并在生理温度下表现出最佳性能[5-7]。但是，PON1的生理作用尚未得到明确鉴定。它被认为是一种混杂的酶[6]。此外，PON1在人群中是多态性的，不同个体也表达了这种酶的广泛不同[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].因此，拟议的研究不仅将着重于表征PON1在其活性位点的反应机制，而且还包括该站点以外的动态信息，因此重新设计的酶将更加催化有效且可高度表达。所得的计算设计将同时测试和表征在湿lab中。实验生物化学将作为约束信息包括在内，以进一步优化设计的周期。研究建议是理论，实验和计算之间的相互作用，这将促进拟议研究的快速进步。候选人的主要职业目标是成为1级研究机构的终身教授，并拥有一个多学科研究小组，该研究小组从事方法论发展和对环境健康科学和生物技术的应用。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聚集在一起，以推动对蛋白质工程的研究。在这一点上，在她的K01奖学金的研究计划中，她的目标是获得正确的专业知识，以精确定位在该交叉点。在这种情况下，她渴望将自己的计算专业知识在McCammon和Dobson Laboratories开发，并将其应用于该领域著名专家之一贝克教授正在追求的酶设计。公共卫生相关性：引入并应用了新的Rosetta酶设计，以重新设计人类多氧蛋白酶，用作有效的生物库助剂，可隔离商业产品中常见的有毒有毒有机磷酸盐化合物。因此，该提案对环境保护和生物技术具有直接相关的影响。