S. coelicolor P450s: Structure/Function/Engineering

描述（由申请人提供）：链霉菌是已知栖息在全球土壤和海洋生态系统中的细菌。在其生态位内，链霉菌通过产生抗细菌、抗真菌和抗线虫化合物以及其他生物活性化合物，开发了复杂而有效的防御其他生物体的机制，其中许多化合物在人类和兽医学中具有价值。链霉菌还表现出广泛的有机毒物解毒能力。这两种防御系统都涉及细胞色素P450（P450）超家族的单加氧酶，约占链霉菌基因的0.25%。链霉菌属中的许多cDNA3基因家族在链霉菌属之间重叠。但是不同的亚家族和家族是明显的，反映了次级代谢的不同需求。P450的多样性反映了选择压力，允许微妙的不同活动的演变和保留，本项目的长期目标是了解底物结合链霉菌P450的一般特征和不同物种中使用的策略，以重新设计P450一级序列，从而产生不同的天然产物。这些信息将建立一个知识库，我们可以从中考虑生产新的和有效的生物活性化合物。天蓝色链霉菌A3（2）的基因组含有18个CYP基因，并且本申请提出确定从我们的前期工作中选择的这些CYP中的8个的功能，将它们的高分辨率X射线结构与功能相关联，并且通过诱变产生这些CYP的修饰形式以检查改变的生物活性。S. coelicolor是一个特别适合这项研究的系统，因为它的大量次级代谢产物是已知的，它们代表了一组不同的有机分子，而且还因为为研究这个物种建立了简单的分子系统。此外，大多数CYP属于相关酶的小组（相同家族或亚家族）。三个具有重叠专业知识和持续合作的实验室已经联手实现这些目标。完成后，这项研究的CYP从S。coelicolor将为其他链霉菌属的改变奠定基础。通过改良的防御系统可以生产新的抗生素和其他人类和动物医学药物。

DESCRIPTION (provided by applicant): Streptomycetes are bacteria known to inhabit the soil and marine ecosystems worldwide. Within their niches streptomycetes have developed complex and efficient mechanisms of defense against other organisms through the production of anti-bacterial, anti-fungal and anti-nematode compounds and other bioactive compounds, many of which have value in human and veterinary medicine. Streptomycetes also exhibit a broad-based ability to detoxify organic poisons. Both these defense systems involve monooxygenases of the cytochrome P450 (CYP) superfamily representing about 0.25% of streptomycete genes. Many of the CYP gene families overlap between Streptomyces spp. as judged by genomic data, but different subfamilies and families are apparent that reflect the different needs in secondary metabolism. The diversity of P450s reflects the selective pressures allowing subtly different activities to evolve and be retained and a long term goal of this project is to understand general features of substrate-binding to streptomycete P450s and strategies used in different species to redesign P450 primary sequence leading to different natural products. This information will establish a knowledge base from which we can consider the production of new and potent bioactive compounds. The genome of Streptomyces coelicolor A3 (2) contains 18 CYP genes and this current application proposes to establish the function of eight of these CYPs selected from our lead-up work, to correlate their high resolution X-ray structure with function, and to generate modified forms of these CYPs by mutagenesis to examine altered biological activity. S. coelicolor is a particularly good system for this study because a large number of its secondary metabolites are known and they represent a diverse group of organic molecules, but also due to the facile molecular systems established for study of this species. Further, most of the CYPs fall into small groups of related enzymes (same family or subfamily). Three laboratories with overlapping expertise and ongoing collaborations have joined forces to achieve these goals. When complete, this study of CYPs from S. coelicolor will set the stage for alteration of other Streptomyces spp. which through modified defense systems can produce new antibiotics and other drugs for human and animal medicine.