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; 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的结构关系的全面研究，NIKD是一种新特征的Flavoenzyme，它催化了Nikkomycins生物合成的关键步骤。 Nikkomycins是肽基核苷抗生素，可抑制几丁质合酶，并已证明是有效的抗真菌剂对几种重要人类病原体的有效抗真菌剂，并且在农业中易于降解杀虫剂。 免疫功能低下的患者中威胁生命的真菌感染的急剧增加，再加上许多当前许多抗真菌药物的耐药性和毒性的出现，为开发新的和更安全的抗真菌剂创造了强大的动力。 NIKD已被确定为单体肌苷氧化酶家族的新成员，这是一组氧化还原酶，表现出对黄素的共价掺入的要求。 总体目标是对重要的一组抗生素的生物合成，这是黄酮酶催化的反应的机理和范围以及扩展的单体肌氨酸氧化酶家族中扩展的底物特异性差异的演变。 NIKD的结构研究将建立在我们最近在1.75埃轴分辨率下确定分离酶的晶体结构方面的成功。 我们将确定该结构揭示的内源配体，确定可能为生理底物氧化过程中形成的中间体提供模型的游离NIKD和酶复合物的结构。 我们旨在定义，动力学来表征和阐明NIKD及其拟议的生理底物，delta-1-或delta-2-二磷脂-2-羧酸盐（P2C）催化的反应的机制。 该化合物可以以两种互变体形式存在。 亚胺（Delta-1-P2C）是中性pH值的主要互变异符。 更容易氧化的谜（Delta-2-P2C）是碱性pH的主要物种。 我们的初步研究表明，NIKD可能是一种新型的三官能酶，可催化其生理底物的亚胺形式的互变异，然后是两个连续的2电子氧化步骤。 如早期的微生物研究所暗示的那样，这些反应产生了最终产物。 在涉及中间陷阱，快速反应动力学，氘标记的底物，底物类似物的研究中，将寻求针对假定反应的明确证据，这些依赖于机械探针，稳态动力学，氘标记的底物，底物类似物作为机械探针的基质，是机械型的稳态素食和稳态素食。