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

• 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
• 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

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.

项目总结/摘要 随着细菌耐药性的出现，新疾病的鉴定，以及对新药物的需求， 随着药物治疗方法的多样化，我们设计新药的能力正成为一个更加紧迫的优先事项。 天然产品通常可用作人类的治疗剂，但存在诸如副作用和副作用等问题。 生产上的困难可能妨碍它们的成功发展。这项建议旨在促进发展 通过合成生物学方法进行治疗，其中分子的生物合成途径被工程化 以改变产品。D类黄素单加氧酶存在于许多天然产物中 生物合成途径，包括缬氨霉素和柔红霉素的生物合成途径，这两种药用天然药物 产品.通过本研究，我们希望能将这些具有代表性的D类黄素单加氧酶 生物合成途径适合于合成生物学目的的工程化。 这里感兴趣的酶催化步骤是多种天然产物共同的生物合成步骤 - 伯胺的黄素依赖性羟基化。在这两种酶中负责这一步骤的酶 生物合成途径-vlmH和DnmZ，分别-将使用瞬时- 状态动力学活性位点残基的定点诱变将与酶活性结合， 结合研究，以验证机械步骤和底物结合相互作用。变动影响 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 改变酶的底物特异性。产生的数据将使vlmH的合理设计成为可能， DnmZ来改变它们的底物结合偏好。类似的研究也可以应用于其他酶。 将多样性引入分子最终结构的途径。