S. coelicolor P450s: Structure/Function/Engineering

描述(申请人提供)：天蓝色链霉菌是典型的链霉菌属。其生理和遗传特性已被广泛研究。该土壤细菌基因组含有18个细胞色素P450(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。这种单加氧酶系统的复合体在次生代谢物的生物合成中扮演着重要的角色，次生代谢物是天蓝色链霉菌中发挥多种不同作用的有机分子，特别是对环境中的有毒生物和化合物具有保护作用。这些化合物中有许多已知具有生物医学活性，并用于治疗人类疾病。在这种链霉菌和其他链霉菌中，P450对初级代谢不是必需的，但在氧化性上定制影响效力和特异性的次级代谢物。这些P450的工程可以导致新的化合物具有新的生物医学能力，该项目的目标是开发一种产生新代谢物的生化范例。在目前的授权期内，我们开发了必要的技术，并获得了初步数据，这将使我们能够在实现这一目标方面向前迈进。该项目涉及范德比尔特的两个实验室(沃特曼和根格里奇)和威尔士大学的两个实验室，现在的斯旺西大学(凯利和兰姆)，以及三个具体目标。(1)链霉菌P450s的功能的确定-将使用底物的预测，基于在细胞中具有已知活性的操纵子中CYP基因的位置，或基于初级序列与已知功能的P450的相似性，并使用敲除菌株来分析与野生型菌株相比的代谢物图谱的差异。这些差异是使用复杂的GC-MS和LC-MS技术来测量的。(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上进行CYP X射线结构测定，以表征底物如何与活性中心结合。(3)天蓝色链霉菌中新次生代谢物的产生--将我们对CYP功能的认识与对CYP结构的认识结合起来，我们将通过定点突变、产物分析、突变酶在天蓝色链霉菌中的表达以及额外的结构确定(如有必要)来产生新的次生代谢物。在当前资助期的40个月里，我们惊讶地发现了这些P450的几个新特征(在中描述)，这些特征扩大了我们对这个单加氧酶超家族的多样性的理解。作为目标2的一部分，我们将进一步研究这些性质。这三个特定目标的意义在于，它们将为设计具有重要生物医学意义的新型链霉菌次生代谢物开发一种范例，并将为细胞色素P450的结构/功能提供新的见解。与公共健康相关：这一竞争性更新的“天蓝色S.coelicolor P450：结构/功能/工程”将继续在一种具有良好特性的土壤细菌中进行P450的研究，长期目标是开发一种使用这些单加氧酶的修饰来产生新的生物医学相关次生代谢物的范例。放线菌(包括天蓝色放线菌)产生大量不同的次生代谢物，其中许多具有生物医学意义。在许多情况下，P450在其生物合成途径的末端以氧化方式定制这些分子，并且P450的修饰可以导致具有新的效力和特异性的新化合物。

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.