Cytochrome P450 Enzymes from Streptomyces: Diverse Biocatalysts in Natural Products Biosynthesis

项目摘要/摘要 研究。这个K99/R00项目的目标是测试细胞色素P450来自 放线菌，一种不同的生物催化剂亚家族，可以作为一种手段来发现自然 具有新功能的产品。我们建议(目标1)研究序列结构函数 来自选定的天然产物生物合成途径的新型P450的关系，(目标2)使用聚焦于P450的 用基因组挖掘的方法发现经历了独特P450催化的天然产物 转换，和(目标3)使用新发现的P450及其同系物作为P450的模型 酶学和生物工程。这种多学科方法包括体外生物信息学分析。 酶学、蛋白质X射线结晶学、体内途径工程、(非)天然产物分离和 结构性决定。出色的初步数据既支持拟议的导师计划的可行性 在两年的时间跨度内分阶段进行，并成功过渡到独立研究计划。这个 这个应用程序的创新之处在于利用了来自专有的P450天然变体的多样性 收集放线菌菌株以发现注入天然产物的前所未有的化学物质 新功能。该项目的意义在于P450催化和酶学的进展和 新天然产物的发现，为未来人类疾病的治疗奠定了基础。 候选人和环境。鲁道夫博士接受了机械、酶和有机方面的研究生培训 由犹他大学杰出的萜类生物化学家C.Dale Poulter博士合成。然后他 与天然产物发现和生物合成领域公认的领导者沈本博士一起在斯克里普斯 ，并接受了放线菌遗传学和天然产物方面的博士后培训 生物合成。他的长期职业目标包括建立一个独立的、有竞争力的研究项目 强调使用天然产物作为发现和研究新化学物质的灵感， 酶学和生物学。他的直接职业目标包括接受P450酶学培训和 结构生物学，以及利用基因组学发现新的酶和天然产物。职业生涯 发展规划。这项提议旨在通过提供以下方式确保他实现职业目标 在指导阶段进行智力、技术和专业培训，这将导致有效 过渡到独立的、终身教职的职位。主要导师沈博士和共同导师乔治博士 菲利普斯是一位X射线结晶学和血红素蛋白质方面的专家，他被选中是因为他们的科学专长和 指导经验。顾问委员会，包括布拉德利·摩尔博士、威尔弗雷德·范德东克博士、肖恩 布雷迪和迈克尔·斯曼斯基都是天然产品方面的专家，他们被明确召集起来监督这两个 这项建议涉及科学和职业发展方面的问题。研究培训和优秀的指导将 TSRI有许多科学和专业发展的机会作为补充。

Project Summary/Abstract Research. The goal of this K99/R00 project is to test the hypothesis that cytochromes P450 from Actinomycetales, a diverse subfamily of remarkable biocatalysts, may be used as a means to discover natural products with novel functionalities. We propose to (Aim 1) investigate the sequence–structure–function relationships of novel P450s from selected natural product biosynthetic pathways, (Aim 2) use a P450-focused genome mining approach to discover natural products that have undergone unique P450-catalyzed transformations, and (Aim 3) employ the newly identified P450s, and their homologues, as models for P450 enzymology and bioengineering. The multidisciplinary approach involves bioinformatics analysis, in vitro enzymology, protein X-ray crystallography, in vivo pathway engineering, (un)natural product isolation and structural determination. Superb preliminary data supports both the feasibility of the proposed mentored phase in a two-year time span, and a successful transition to an independent research program. The innovation of this application lies in leveraging the diversity of natural P450 variants from a proprietary Actinomycetales strain collection for discovering unprecedented chemistries that imbue natural products with new functionalities. The significance of this project is the advancement of P450 catalysis and enzymology and the discovery of novel natural products, laying the foundation for future treatments of human diseases. Candidate and Environment. Dr. Rudolf obtained graduate training in mechanistic enzymology and organic synthesis from Dr. C. Dale Poulter, a preeminent terpenoid biochemist, at the University of Utah. He then joined Dr. Ben Shen, a recognized leader in natural products discovery and biosynthesis, at The Scripps Research Institute (TSRI), and received postdoctoral training in actinomycetes genetics and natural products biosynthesis. His long-term career goals include establishing an independent, competitive research program that emphasizes using natural products as inspirations for the discovery and study of novel chemistries, enzymologies, and biologies. His immediate career goals include obtaining training in P450 enzymology and structural biology, and in utilizing genomics for the discovery of novel enzymes and natural products. Career development plan. This proposal was designed to ensure the realization of his career goals by providing intellectual, technical, and professional training during the mentored phase that will lead to an effective transition to an independent, tenure-track position. The primary mentor, Dr. Shen, and co-mentor, Dr. George Phillips, an expert in X-ray crystallography and heme proteins, were chosen for their scientific expertise and mentoring experience. An advisory committee, including Drs. Bradley Moore, Wilfred van der Donk, Sean Brady, and Michael Smanski, all experts in natural products, was explicitly assembled to oversee both the science and career development aspects of this proposal. The research training and excellent mentoring will be supplemented with numerous opportunities for scientific and professional development available at TSRI.