Studies on NikD, a Nikkomycin Biosynthetic Enzyme

尼可霉素生物合成酶NikD的研究

• 批准号: 7012788
• 负责人: MARILYN S JORNS
• 金额: $ 24.9万
• 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/7012788
• 关键词: antifungal antibiotics; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate; flavins; flavoproteins; microorganism metabolism; oxidation reduction reaction; protein engineering; stop flow technique; structural biology; 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结构与功能的关系的全面调查，nikD是一种新鉴定的黄素酶，催化Nikmycins生物合成的关键步骤。尼可霉素是一种抑制甲壳素合成酶的肽基核苷类抗生素，在农业上已被证明是有效的抗真菌药物，可有效对抗多种重要的人类病原体和易降解的杀虫剂。在免疫功能低下的患者中，危及生命的真菌感染急剧增加，再加上目前许多抗真菌药物出现耐药性和毒性，为开发新的、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，这是一组氧化还原酶，需要黄素的共价结合。总的目标是更深入地了解一类重要抗生素的生物合成，黄素酶催化反应的机制和范围，以及扩大的单体肌氨酸氧化酶家族内底物特异性差异的演变。与nikD的结构研究将建立在我们最近在1.75埃分辨率下确定分离酶的晶体结构的成功的基础上。我们将鉴定这种结构揭示的内源配体，确定游离NikD和酶复合体的结构，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与其生理底物-β-1-或-β-2-哌啶-2-羧酸盐(P2C)催化的反应(S)的反应机理。这种化合物可以以两种互变异构形式存在。在中性pH下，亚胺(Delta-1-P2C)是主要的互变异构体。在碱性pH条件下，较易被氧化的烯胺(Delta-2-P2C)是主要物种。我们的初步研究表明，nikD可能是一种新的三功能酶，它催化其生理底物亚胺形式的互变异构化，随后是两个连续的2电子氧化步骤。早期的微生物学研究表明，这些反应会产生最终产物吡啶甲酸酯。将在涉及中间捕获、快速反应动力学、氚标记底物、充当机械探针的底物类似物、稳态动力学、氚标记底物、充当机械探针的底物类似物、稳态动力学、诱变和改良黄素衍生物的研究中寻找假定反应的确凿证据。