Engineering isobutylamine N-hydroxylase for applications in antibiotic biosynthes

描述(申请人提供)：随着细菌耐药性的出现，新疾病的发现，以及对不同疗效的新疗法的需求，我们设计抗击细菌感染的药物的能力正成为更紧迫的优先事项。天然产品经常被用作治疗人类的药物，尽管副作用和生产困难等问题可能会阻碍它们的成功开发。这项建议试图通过研究万乃尼霉素生物合成途径中的结构-功能关系来解决对新抗生素的需求。这种自然可得的抗生素对革兰氏阳性和革兰氏阴性细菌有疗效，并显示出一些抗癌治疗的前景。通过这项研究，我们希望使伐拉尼霉素符合一种新的药物开发战略-合成生物学，在合成生物学中，药物的生物合成途径被设计为允许在产品中引入多样性。这里描述的研究集中在多种抗生素共同的生物合成步骤-依赖黄素的伯胺羟基化。负责万乃馨生物合成途径这一步骤的酶将在结构和生化特征上进行表征。这些研究确定的构效关系将通过生物信息学和生化技术来验证，包括结合酶活性和结合研究的诱变。所获得的数据将使合理设计vlmH来改变其底物结合特异性。这样的研究可以在该途径的其他步骤上进行；通过这种方式，我们可以将多样性引入万乃尼霉素的最终结构。如果有足够的时间和研究，这种分子可以开发成一种有用的治疗药物。

DESCRIPTION (provided by applicant): With the emergence of bacterial resistance, identification of new diseases, and the need for new therapeutics with different efficacies, our ability to design drugs to battle bacterial infections is becoming a more urgent priority. Natural products are often useful as therapeutics for humans, though problems such as side effects and production difficulties can preclude their successful development. This proposal seeks to address the need for new antibiotics by studying structure-function relationships in the valanimycin biosynthetic pathway. This naturally available antibiotic has efficacy against gram positive and gram negative bacteria, and shows some promise as an anticancer therapeutic. With this research we hope to make valanimycin amenable to a new drug development strategy, synthetic biology, in which the drug's biosynthetic pathway is engineered to allow introduction of diversity into the product. The research described here focuses on a biosynthetic step common to multiple antibiotics - flavin-dependent hydroxylation of a primary amine. The enzyme responsible for this step in the valanimycin biosynthetic pathway will be structurally and biochemically characterized. The structure activity relationships identified by those studies will be verified by bioinformatics and biochemical techniques, including mutagenesis combined with enzymatic activity and binding studies. The data yielded will enable rational design of vlmH to alter its substrate binding specificity. Such studies can be pursued on other steps of the pathway; in this way, we can introduce diversity into the valanimycin final structure. Given enough time and research, this molecule could be developed into a useful therapeutic.