Mechanistic studies to enable rational design Class D monooxygenases

机制研究以实现合理设计 D 类单加氧酶

基本信息

批准号：
9976539
负责人：
Jessica Vey
金额：
$ 10.52万
依托单位：
CALIFORNIA STATE UNIVERSITY NORTHRIDGE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-15 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9976539
关键词：
Active Sites Address Affect Affinity Amines Amino Acids Antibiotics Bacterial Infections Binding Binding Sites Biochemical Biological Biological Assay Biotechnology Catalysis Complement component C4a Computer software Crystallography Data Daunorubicin Development Disease Dissociation Drug resistance Engineering Enzymes Family Family member Flavins Goals Homologous Gene Human Hydroxylation Kinetics Knowledge Laboratories Methods Mixed Function Oxygenases Modification Mutagenesis Mutation Natural Products Pathway interactions Pharmaceutical Preparations Play Production Property Proteins Reaction Research Resistance Role Sequence Alignment Sequence Homology Site-Directed Mutagenesis Spectrophotometry Structure Structure-Activity Relationship Substrate Specificity Surface Plasmon Resonance Testing Therapeutic Work bacterial resistance base course development design enzyme activity enzyme pathway enzyme substrate experimental study infectious disease treatment interest member novel therapeutics oxidation preference side effect synthetic biology therapeutic development tool valanimycin

项目摘要

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.

项目概要/摘要随着细菌耐药性的出现、新疾病的识别以及对新药物的需求不同功效的治疗方法，我们设计新药的能力正变得更加紧迫。天然产品通常可用作人类的治疗药物，但存在副作用和副作用等问题。生产困难可能会阻碍其成功开发。该提案旨在促进开发。通过合成生物学方法进行治疗，其中分子的生物合成途径被设计为了改变产品，D 类黄素单加氧酶存在于许多天然产品中。生物合成途径，包括缬氨霉素和柔红霉素（两种药用天然化合物）的生物合成途径通过这项研究，我们希望制造出这些具有代表性的 D 类黄素单加氧酶。适合合成生物学目的的生物合成途径。这里感兴趣的酶催化步骤是多种天然产物常见的生物合成步骤 – 伯胺的黄素依赖性羟基化负责这两个步骤的酶。生物合成途径——分别是 vlmH 和 DnmZ——将使用瞬时- 活性位点残基的定点诱变将与酶活性结合起来。结合研究以验证机械步骤和底物结合相互作用的变化的影响。将研究底物结合位点对中间体形成动力学的影响，以用于验证对酶底物特异性的修改所产生的数据将使 vlmH 和 vlmH 的合理设计成为可能。 DnmZ 改变其底物结合偏好，类似的研究可以应用于其他酶。将多样性引入分子最终结构的途径。