S. coelicolor P450s: Structure/Function/Engineering

S. coelicolor P450s：结构/功能/工程

• 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
• 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

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（由申请方提供）：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。该菌基因组中含有18个细胞色素P450（CYP 450）基因、6个铁氧还蛋白基因和4个铁氧还蛋白还原酶基因。这种单加氧酶系统的复合体在次生代谢物的生物合成中起着重要作用，次生代谢物是在S.腔棘鱼，特别是对环境中有毒生物和化合物的保护。已知这些化合物中的许多具有生物医学活性并用于治疗人类疾病。P450对这种和其他链霉菌的初级代谢不是必需的，但氧化调节次级代谢产物，影响效价和特异性。这些P450的工程可以导致具有新的生物医学能力的新化合物，该项目的目标是开发用于产生新代谢物的生物化学范例。在目前的资助期内，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这一目标。该项目涉及两个实验室在范德比尔特（沃特曼和Guengerich）和两个在威尔士大学，现在斯旺西大学（凯利和兰姆），和三个具体的目标。(1)链霉菌P450 s功能的测定-将使用基于在细胞中具有已知活性的操纵子中的p53基因的位置或基于一级序列与已知功能的P450 s的相似性的底物预测，以及使用敲除菌株分析与野生型菌株相比代谢物谱的差异来进行。这些差异使用复杂的GC-MS和LC-MS技术测量。(2)链霉菌P450的结构测定和新型P450性质的生物物理研究-我们将主要集中在具有已知功能的P450上的X射线结构测定，以表征底物如何结合在活性位点。(3)S.中新次级代谢产物的产生。coelicolor -将我们对α-淀粉酶功能的理解与α-淀粉酶结构的理解结合起来，我们将依靠定点诱变、产物分析、突变酶在S. coelicolor和其他结构测定（如有必要）。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（如内所述），这些特征扩展了我们对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属性。这三个特定目标的意义在于，它们将为工程化具有重要的新生物医学相关性潜力的新型链霉素次级代谢物开发一种范例，并将为细胞色素P450结构/功能提供新的见解。公共卫生相关性：这一竞争性的“S。天蓝色P450：“结构/功能/工程”将继续在一个良好表征的土壤细菌中进行P450的研究，其长期目标是开发一个使用这些单加氧酶的修饰来产生新的生物医学相关次级代谢产物的范例。放线菌（包括S. coelicolor）产生大量不同的次级代谢物，其中许多具有生物医学相关性。在许多情况下，P450在其生物合成途径的末端附近氧化地定制这些分子，并且P450的修饰可以产生具有新的效力和特异性的新化合物。