Engineering isobutylamine N-hydroxylase for applications in antibiotic biosynthes

描述（由申请人提供）：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，我们设计对抗细菌感染的药物的能力变得更加紧迫。天然产物通常作为人类的治疗药物有用，尽管副作用和生产困难等问题可能阻碍它们的成功开发。本研究旨在通过研究瓦尼霉素生物合成途径的结构-功能关系来解决对新抗生素的需求。这种天然可用的抗生素对革兰氏阳性和革兰氏阴性细菌都有效，并显示出抗癌治疗的一些希望。通过这项研究，我们希望使瓦尼霉素适应一种新的药物开发策略——合成生物学，在这种策略中，药物的生物合成途径被设计成允许在产品中引入多样性。这里描述的研究重点是多种抗生素共同的生物合成步骤-黄素依赖伯胺的羟基化。在valanimycin生物合成途径中负责这一步的酶将被结构和生物化学表征。这些研究确定的结构活性关系将通过生物信息学和生化技术进行验证，包括诱变与酶活性和结合研究相结合。所得数据将有助于合理设计vlmH以改变其底物结合特异性。这种研究可以在途径的其他步骤上进行；这样，我们就可以将多样性引入到瓦尼霉素的最终结构中。如果有足够的时间和研究，这种分子可以发展成一种有用的治疗方法。

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.