Mechanistic studies to enable rational design Class D monooxygenases

项目摘要/摘要 随着细菌耐药性的出现，新疾病的鉴定，以及对新的 随着治疗效果的不同，我们设计新药的能力正成为更紧迫的优先事项。 天然产品通常被用作治疗人类的药物，尽管诸如副作用和 生产困难可能会阻碍它们的成功开发。这项提议旨在使发展 通过合成生物学方法进行治疗，其中分子的生物合成途径是通过工程设计的 为了改变产品。D类黄素单加氧酶存在于多种天然产物中 生物合成途径，包括万乃馨和柔红霉素这两种药用天然药物的合成途径 产品。通过这项研究，我们希望使这些具有代表性的D类黄素单加氧酶 可用于合成生物学目的的工程生物合成途径。 这里所涉及的酶催化步骤是多种天然产物共有的生物合成步骤 -伯胺的黄素依赖羟基化。在这两个步骤中负责这一步的酶 生物合成途径-分别为vlmH和DnmZ-将使用瞬变- 状态动力学。活性位点残基的定点突变将与酶活性和 结合研究，以验证机械步骤和底物结合相互作用。这一变化的影响 将研究底物结合部位对中间体形成动力学的影响，以用于验证 对酶的底物专一性的修饰。所得数据将使VLMH和VLMH的合理设计成为可能 DNMZ改变它们的底物结合偏好。类似的研究也可以应用于大豆中的其他酶 将多样性引入分子最终结构的途径。

PROJECT SUMMARY / ABSTRACT With the emergence of bacterial resistance, identification of new diseases, and the need for new therapeutics with different efficacies, our ability to design new drugs 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 enable development of therapeutics through synthetic biology methods, in which the molecule's biosynthetic pathway is engineered in order to alter the product. The Class D flavin monooxygenases are found in numerous natural product biosynthetic pathways, including those of valanimycin and daunorubicin, two medicinally useful natural products. With this research we hope to make the Class D flavin monooxygenases of these representative biosynthetic pathways amenable to engineering for synthetic biology purposes. The enzyme-catalyzed step of interest here is a biosynthetic step common to multiple natural products – flavin-dependent hydroxylation of a primary amine. The enzymes responsible for this step in the two biosynthetic pathways – vlmH and DnmZ, respectively – will be biochemically characterized using transient- state kinetics. Site-directed mutagenesis of active site residues will be combined with enzymatic activity and binding studies to validate mechanistic steps and substrate binding interactions. The effect of changes in the substrate binding site on the kinetics of intermediate formation will be investigated for use in validating modifications to the enzyme's substrate specificity. The data yielded will enable rational design of vlmH and DnmZ to alter their substrate binding preferences. Similar studies can be applied to other enzymes of the pathways to introduce diversity into the molecules' final structures.