Studies on NikD, a Nikkomycin Biosynthetic Enzyme

尼可霉素生物合成酶NikD的研究

• 批准号: 7169841
• 负责人: MARILYN S JORNS
• 金额: $ 24.42万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169841
• 关键词: Active Sites; Aerobic; Agriculture; Amino Acids; Anabolism; Antibiotics; Antifungal Agents; Antifungal Antibiotics; Binding; Biochemical; Catalysis; Chitin Synthase; Condition; Coupled; Data; Depth; Deuterium; Development; Drug resistance; Electrons; Enzymes; Evolution; Exhibits; Family; Flavins; Goals; Histidine; Human; Hydrogen Peroxide; Imines; Immunocompromised Host; In Vitro; Insecticides; Investigation; Kinetics; Label; Life; Ligands; Modeling; Molecular; Monitor; Movement; Multienzyme Complexes; Mutagenesis; Mutate; Mycoses; Nature; Nucleosides; Oxidants; Oxidation-Reduction; Oxygen; Pathway interactions; Physiological; Positioning Attribute; Property; Rate; Reaction; Relative (related person); Research; Research Personnel; Resolution; Role; Sarcosine oxidase; Series; Site; Solutions; Structure; Substrate Specificity; Thinking; Toxic effect; analog; base; carboxylate; electron donor; member; mutant; nikkomycin; novel; oxidation; pathogen; picolinate; programs; research study; success; tautomer

DESCRIPTION (Provided by the applicant): The proposed research involves a comprehensive investigation of the relationship of structure to function in nikD, a newly characterized flavoenzyme that catalyzes a key step in the biosynthesis of nikkomycins. Nikkomycins are peptidyl nucleoside antibiotics that inhibit chitin synthase and have proven effective as potent antifungal agents against several important human pathogens and easily degraded insecticides in agriculture. The dramatic increase of life-threatening fungal infections in immunocompromised patients, coupled with the emergence of drug resistance and toxicity of many current antifungal drugs, has created a strong impetus for the development of new and safer antifungal agents. NikD has been identified as a new member of the monomeric sarcosine oxidase family, a group of redox enzymes that exhibit a requirement for covalent incorporation of flavin. The overall goal is to gain a deeper understanding of the biosynthesis of an important group of antibiotics, the mechanism and scope of reactions catalyzed by flavoenzymes and the evolution of substrate specificity differences within the expanding monomeric sarcosine oxidase family. Structural studies with nikD will build on our recent success in determining the crystal structure of the isolated enzyme at 1.75 Angstroms resolution. We will identify the endogenous ligand revealed by this structure, determine the structure of free nikD and enzyme complexes likely to provide models for intermediates formed during oxidation of the physiological substrate. We aim to define, kinetically characterize and elucidate the mechanism of the reactions(s) catalyzed by nikD with its proposed physiological substrate, delta-1- or delta-2-piperideine-2-carboxylate (P2C). This compound can exist in two tautomeric forms. The imine (delta-1-P2C) is the predominant tautomer at neutral pH. The more easily oxidized enamine (delta-2-P2C) is the major species at alkaline pH. Our preliminary studies suggest that nikD may be a novel trifunctional enzyme that catalyzes tautomerization of the imine form of its physiological substrate, followed by two successive 2-electron oxidation steps. These reactions yield picolinate as the final product, as suggested by earlier microbiological studies. Definitive evidence for the postulated reactions will be sought in studies that involve intermediate trapping, rapid reaction kinetics, deuterium-labeled substrate, substrate analogs that act as mechanistic probes, steady-state kinetics, deuterium-labeled substrate, substrate analogs that act as mechanistic probes, steady-state kinetics, mutagenesis and modified flavin derivatives.

描述（由申请人提供）：拟议的研究涉及对 nikD 结构与功能关系的全面研究，nikD 是一种新表征的黄素酶，可催化尼可霉素生物合成的关键步骤。 尼可霉素是肽基核苷抗生素，可抑制几丁质合酶，并已被证明可作为有效的抗真菌剂，对抗几种重要的人类病原体和农业中易降解的杀虫剂。 免疫功能低下患者中危及生命的真菌感染急剧增加，加上当前许多抗真菌药物出现耐药性和毒性，为开发新型、更安全的抗真菌药物创造了强大的动力。 NikD 已被确定为单体肌氨酸氧化酶家族的新成员，该家族是一组需要共价结合黄素的氧化还原酶。 总体目标是更深入地了解一组重要抗生素的生物合成、黄素酶催化反应的机制和范围以及不断扩大的单体肌氨酸氧化酶家族内底物特异性差异的演变。 nikD 的结构研究将建立在我们最近成功确定分离酶的晶体结构（分辨率为 1.75 埃）的基础上。 我们将鉴定该结构揭示的内源配体，确定游离 nikD 和酶复合物的结构，这可能为生理底物氧化过程中形成的中间体提供模型。 我们的目标是定义、动力学表征和阐明 nikD 与其提出的生理底物 delta-1- 或 delta-2-哌啶-2-羧酸酯 (P2C) 催化的反应机制。 该化合物可以两种互变异构形式存在。 亚胺 (delta-1-P2C) 是中性 pH 条件下主要的互变异构体。 更容易氧化的烯胺 (delta-2-P2C) 是碱性 pH 值下的主要物质。 我们的初步研究表明，nikD 可能是一种新型三功能酶，可催化其生理底物亚胺形式的互变异构化，然后进行两个连续的 2 电子氧化步骤。 正如早期微生物学研究表明的那样，这些反应产生吡啶甲酸作为最终产物。 将在涉及中间捕获、快速反应动力学、氘标记底物、充当机械探针的底物类似物、稳态动力学、氘标记底物、充当机械探针的底物类似物、稳态动力学、诱变和改性黄素衍生物的研究中寻找假定反应的确定证据。