Studies on NikD, a Nikkomycin Biosynthetic Enzyme

描述（由申请人提供）：拟议的研究涉及对nikD的结构与功能关系的全面研究，nikD是一种新发现的黄酶，催化nikkomycin生物合成的关键步骤。nikkomycin是一种抑制几丁质合成酶的肽基核苷类抗生素，已被证明是一种有效的抗真菌剂，可以对抗几种重要的人类病原体和农业中容易降解的杀虫剂。免疫功能低下患者中危及生命的真菌感染的急剧增加，加上许多现有抗真菌药物的耐药性和毒性的出现，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，这是一组需要黄素共价结合的氧化还原酶。总体目标是更深入地了解一类重要抗生素的生物合成，黄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中底物特异性差异的演变。利用nikD进行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晶体结构的成功基础上。我们将鉴定这种结构揭示的内源性配体，确定游离nikD和酶复合物的结构，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与其提出的生理底物，δ -1-或δ -2-哌啶-2-羧酸盐（P2C）催化的反应机制。这种化合物能以两种互变异构形式存在。在中性ph下，亚胺（δ -1- p2c）是主要的互变异构体。在碱性ph下，更容易被氧化的烯胺（δ -2- p2c）是主要的互变异构体。我们的初步研究表明，nikD可能是一种新型的三功能酶，它催化亚胺形式的生理底物的互变异构体，随后是两个连续的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盐作为最终产物。将在涉及中间捕获、快速反应动力学、氘标记的底物、作为机械探针的底物类似物、稳态动力学、氘标记的底物、作为机械探针的底物类似物、稳态动力学、诱变和修饰的黄素衍生物的研究中寻找假设反应的明确证据。

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.