Studies on NikD, a Nikkomycin Biosynthetic Enzyme

尼可霉素生物合成酶NikD的研究

• 批准号: 7340410
• 负责人: MARILYN S JORNS
• 金额: $ 23.95万
• 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 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7340410
• 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（由申请方提供）：拟定研究涉及nikD结构与功能关系的全面研究，nikD是一种新表征的黄素酶，催化尼可霉素生物合成的关键步骤。 尼可霉素是抑制几丁质合成酶的肽基核苷类抗生素，已被证明是有效的抗真菌剂，可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的急剧增加，加上目前许多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已被鉴定为单体肌氨酸氧化酶家族的新成员，该家族是一组表现出需要共价结合黄素的氧化还原酶。 总的目标是获得一个重要的抗生素组的生物合成，由flavoenzymes催化的反应的机制和范围和底物特异性差异的不断扩大的单体肌氨酸氧化酶家族内的演变更深入的了解。 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的晶体结构的成功基础上。 我们将确定这种结构所揭示的内源性配体，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。 我们的目的是定义，动力学表征和阐明的反应（S）催化的nikD与其提出的生理底物，δ-1-或δ-2-哌啶-2-羧酸（P2C）的机制。 该化合物可以以两种互变异构形式存在。 亚胺（delta-1-P2C）是主要的互变异构体在中性pH值。更容易氧化的烯胺（delta-2-P2C）是在碱性pH值的主要物种。我们的初步研究表明，nikD可能是一种新的三功能酶，催化其生理底物的亚胺形式的互变异构化，随后通过两个连续的2-电子氧化步骤。 这些反应产生吡啶甲酸酯作为最终产物，如早期的微生物学研究所建议的。 假设的反应的证据将寻求在研究中，涉及中间体捕获，快速反应动力学，氘标记的基板，基板类似物，作为机械探针，稳态动力学，氘标记的基板，基板类似物，作为机械探针，稳态动力学，诱变和改性黄素衍生物。

项目成果

A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis.

移动色氨酸是尼可霉素抗生素生物合成所必需的黄素酶中的内在电荷转移供体。

• DOI: 10.1021/bi062087s
• 发表时间: 2007
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Bruckner,RobertC;Zhao,Gouhua;Ferreira,Patricia;Jorns,MarilynSchuman
• 通讯作者: Jorns,MarilynSchuman

Probing the role of active site residues in NikD, an unusual amino acid oxidase that catalyzes an aromatization reaction important in nikkomycin biosynthesis.

探究 NikD 中活性位点残基的作用，NikD 是一种不寻常的氨基酸氧化酶，可催化尼可霉素生物合成中重要的芳香化反应。

• DOI: 10.1021/bi9006918
• 发表时间: 2009
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Kommoju,Phaneeswara-Rao;Bruckner,RobertC;Ferreira,Patricia;Jorns,MarilynSchuman
• 通讯作者: Jorns,MarilynSchuman