S. coelicolor P450s: Structure/Function/Engineering

描述（由申请人提供）：链霉菌是已知栖息在世界各地土壤和海洋生态系统中的细菌。在它们的生态位中，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，发展出了复杂而有效的防御其他生物的机制，其中许多化合物在人类和兽药中具有价值。链霉菌还表现出广泛的解毒有机毒物的能力。这两种防御系统都涉及细胞色素P450 （CYP）超家族的单加氧酶，约占链霉菌基因的0.25%。从基因组数据判断，链霉菌之间的CYP基因家族有许多重叠，但不同的亚家族和家族反映了次生代谢的不同需求。P450的多样性反映了选择压力，使得微妙的不同活动得以进化和保留，该项目的长期目标是了解底物与链霉菌P450结合的一般特征，以及在不同物种中重新设计P450初级序列从而产生不同天然产物的策略。这些信息将建立一个知识库，从中我们可以考虑生产新的和有效的生物活性化合物。coelicolor链霉菌A3(2)的基因组包含18个CYP基因，目前的应用建议建立从我们的前期工作中选择的8个CYP基因的功能，将它们的高分辨率x射线结构与功能联系起来，并通过诱变产生这些CYP的修饰形式来检测改变的生物活性。S. coelicolor是本研究的一个特别好的系统，因为它的大量次生代谢物是已知的，它们代表了多样化的有机分子群，而且还因为为研究该物种建立了简单的分子系统。此外，大多数CYPs属于相关酶的小群体（同一家族或亚家族）。具有重叠专业知识和正在进行的合作的三个实验室已联手实现这些目标。该研究完成后，将为其他链霉菌的改造奠定基础，通过改造防御系统，可以生产出新的抗生素和其他人畜药物。

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.