S. coelicolor P450s: Structure/Function/Engineering

S. coelicolor P450s：结构/功能/工程

基本信息

批准号：
7527481
负责人：
F PETER Guengerich
金额：
$ 43.84万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7527481
关键词：
Actinobacteria class Actinomyces Active Sites Address Anabolism Antibiotics Antineoplastic Agents Arts Bacteria Binding Biochemical Biological Process Cells Chemicals Complex Crystallography Cytochrome P450 Cytochromes Data Engineering Environment Enzymes Ferredoxin Funding Generations Genes Genetic Genome Goals Grant Growth Investigation Knock-out Laboratories Lead Link Location Marines Measures Metabolic Metabolic Biotransformation Metabolism Mixed Function Oxygenases Modification Numbers Operon Organism Orphan Oxidoreductase Pathway interactions Pharmaceutical Preparations Physiological Play Poison Production Progress Reports Property Public Health Resolution Roentgen Rays Role Role playing therapy Site-Directed Mutagenesis Soil Specificity Streptomyces Streptomyces coelicolor Structure System Technology Thinking Universities Wales Work base human disease insight microorganism mutant novel polyketide synthase wasting

项目摘要

DESCRIPTION (provided by applicant): Streptomyces coelicolor is the prototypic Streptomycete spp. Its physiological and genetic properties have been studied extensively. The genome of this soil bacterium contains 18 cytochrome P450s (CYP) genes, 6 ferredoxin genes and 4 ferredoxin reductase genes. This complex of monooxygenase systems plays important roles in the biosynthesis of secondary metabolites, organic molecules which serve several different roles in S. coelicolor, particularly protection against toxic organisms and compounds in the environment. Many of these compounds are known to have biomedical activity and are used in treatment of human disease. The P450s are not essential for primary metabolism in this and other streptomycetes but oxidatively tailor secondary metabolites influencing both potency and specificity. Engineering of these P450s can lead to novel compounds having new biomedical capacity and the goal of this project is to develop a biochemical paradigm for generating new metabolites. During the current granting period we have developed the necessary technology and obtained preliminary data which will permit us to move aggressively forward in addressing this goal. This project involves two laboratories at Vanderbilt (Waterman and Guengerich) and two at the University of Wales, now Swansea University (Kelly and Lamb), and three Specific Aims. (1) Determination of Function of Streptomyces P450s - will be carried out using prediction of substrate based on location of CYP genes in operons having known activity in cells, or based on similarity of primary sequence with P450s of known function, and use of knockout strains for analysis of differences in metabolite profiles compared to the wild type strain. These differences are measured using sophisticated GC-MS and LC-MS technologies. (2) Structure Determination of Streptomyces P450s and Biophysical Investigation of Novel P450 Properties -we will focus our effort of CYP X-ray structure determination primarily on P450s having known functions to characterize how substrates bind in the active sites. (3) Generation of Novel Secondary Metabolites in S. coelicolor - putting together our understanding of CYP function with that of CYP structure we will generate novel secondary metabolites relying on site directed mutagenesis, product analysis, expression of mutant enzymes in S. coelicolor, and additional structure determination (if necessary). During the 40 months of the current funding period we have been surprised to find several novel features of these P450s (described within) which expand our understanding of the diversity of this superfamily of monooxygenases. As part of Aim 2 we will further investigate these properties. The significance of these three Specific Aims is that they will develop a paradigm for engineering novel streptomyces secondary metabolites with the potential of important new biomedical relevance and will provide new insights into cytochrome P450 structure/function. PUBLIC HEALTH RELEVANCE: This competing renewal of "S. coelicolor P450s: Structure/Function/Engineering" will continue ongoing studies of P450s in a well characterized soil bacterium with the long term goal to develop a paradigm for using modification of these monooxygenases to generate novel biomedically relevant secondary metabolites. Actinomycetes (including S. coelicolor) produce a large number of different secondary metabolites many of which have biomedical relevance. In many cases P450s oxidatively tailor these molecules near the end of their biosynthetic pathways and modification of P450s can lead to new compounds having novel potency and specificity.

描述（由申请人提供）：天蓝色链霉菌是原型链霉菌属。其生理和遗传特性已被广泛研究。该土壤细菌的基因组包含18个细胞色素P450s (CYP)基因、6个铁氧还蛋白基因和4个铁氧还蛋白还原酶基因。这种单加氧酶系统复合物在次生代谢物和有机分子的生物合成中发挥着重要作用，这些有机分子在天蓝色链球菌中发挥着多种不同的作用，特别是针对环境中的有毒生物体和化合物的保护。已知许多这些化合物具有生物医学活性并用于治疗人类疾病。 P450 对于这种链霉菌和其他链霉菌的初级代谢来说并不是必需的，但会氧化调整影响效力和特异性的次级代谢产物。这些 P450 的工程可以产生具有新生物医学能力的新型化合物，该项目的目标是开发用于生成新代谢物的生化范例。在当前的资助期内，我们已经开发了必要的技术并获得了初步数据，这将使我们能够积极推进实现这一目标。该项目涉及范德比尔特大学的两个实验室（Waterman 和 Guengerich）和威尔士大学（现为斯旺西大学）的两个实验室（Kelly 和 Lamb）以及三个具体目标。 (1) 链霉菌 P450 功能的确定 - 将使用基于细胞中具有已知活性的操纵子中 CYP 基因的位置的底物预测，或基于与已知功能的 P450 的一级序列的相似性，以及使用敲除菌株来进行用于分析与野生型菌株相比代谢物谱的差异。这些差异是使用先进的 GC-MS 和 LC-MS 技术测量的。 (2) 链霉菌 P450 的结构测定和 P450 新特性的生物物理研究 - 我们将主要将 CYP X 射线结构测定的工作重点放在具有已知功能的 P450 上，以表征底物如何在活性位点中结合。 (3) 在 S. coelicolor 中产生新的次生代谢物 - 将我们对 CYP 功能的理解与 CYP 结构的理解结合起来，我们将依靠定点诱变、产物分析、S. coelicolor 中突变酶的表达来产生新的次生代谢物，以及额外的结构确定（如果需要）。在当前资助期的 40 个月内，我们惊讶地发现这些 P450 的几个新特征（见下文所述），这些特征扩展了我们对这个单加氧酶超家族多样性的理解。作为目标 2 的一部分，我们将进一步研究这些特性。这三个具体目标的意义在于，它们将开发一种工程新型链霉菌次生代谢物的范例，具有重要的新生物医学相关性的潜力，并将为细胞色素 P450 结构/功能提供新的见解。公共健康相关性：“S. coelicolor P450s：结构/功能/工程”的竞争性更新将继续对一种已充分表征的土壤细菌中的 P450s 进行研究，长期目标是开发一个范例，利用这些单加氧酶的修饰来产生新的单加氧酶。生物医学相关的次级代谢产物。放线菌（包括天蓝色链球菌）产生大量不同的次生代谢物，其中许多具有生物医学相关性。在许多情况下，P450 在接近其生物合成途径末端时会氧化调整这些分子，并且 P450 的修饰可以产生具有新效力和特异性的新化合物。