Discovery and Characterization of Novel Halogenases from the Human Microbiome

来自人类微生物组的新型卤化酶的发现和表征

• 批准号: 10360052
• 负责人: John J. Bellizzi
• 金额: $ 42.47万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360052
• 关键词: Agrochemicals; Aromatic Compounds; Bacteria; Biochemistry; Bioinformatics; Brain; Bromine; Cells; Chemicals; Chemistry; Communication; Communities; Coupling; Crystallization; Data; Enzymatic Biochemistry; Enzymes; Escherichia coli; Exhibits; Family member; Flavins; Gastrointestinal tract structure; Genes; Goals; Human; Human Microbiome; Immunity; Indoles; Investigation; Iodination reaction; Metabolism; Microbe; Open Reading Frames; Organic Synthesis; Outcome; Pathway interactions; Pharmacologic Substance; Physiological Processes; Play; Preparation; Process; Prokaryotic Cells; Proteins; Reaction; Recombinants; Research; Research Personnel; Roentgen Rays; Role; Sampling; Shotgun Sequencing; Skin; Soil; Structure; Students; System; Time; Tissues; Training Programs; Transition Elements; Tryptophan; X ray diffraction analysis; aryl halide; base; bioinformatics tool; chlorination; cohort; data mining; design; exhaustion; experimental study; fungus; gene product; halogenation; human tissue; improved; insight; interest; member; metagenome; microbial; microbiome; microorganism; novel; public health relevance; scaffold; structural biology; undergraduate student

PROJECT SUMMARY/ABSTRACT Flavin-dependent halogenases (FDHs) are biosynthetic enzymes from bacteria and fungi that catalyze regioselective C-X bond formation (X=Cl, Br, I) with various aromatic substrates. Their regioselectivity and environmentally friendly reaction conditions make FDHs attractive candidates for green synthesis of aryl halides to be used in transition metal catalyzed cross- coupling reactions. The proposed project will expand our understanding of regioselective aryl halide formation by FDHs through the identification and structural and enzymatic characterization of novel members of this family of enzymes, expanding the FDH toolkit by adding new members with different substrate and halide selectivities to the currently characterized enzymes. In Aim 1 of this project, we will fully characterize the extended substrate scope of AbeH and BorH, two FDHs from soil bacteria metagenomes that we have shown can chlorinate and brominate tryptophan to produce two different regioisomers. Aims 2 and 3 are based on our hypothesis that FDHs are also produced by the bacteria of the human microbiome, where they could potentially generate secondary metabolites that play a role in chemical communication between bacteria and human cells. This hypothesis is supported by the fact that a simple bioinformatic search of three sequence files from human microbiome metagenome shotgun sequencing projects yielded five ORFs predicted to encode novel FDHs. Preliminary investigations of the first of the five hypothetical FDH genes has verified that it encodes a protein with the ability to halogenate indole. Aim 2 will involve the full characterization of the enzymatic activity, substrate and halide scope, and crystal structures of these five putative microbiome FDHs. Aim 3 will establish a training program in bioinformatics, enzymology, and structural biology for undergraduate biochemistry majors, who will mine human microbiome metagenome sequences for additional putative FDH genes, express and purify the gene products in E. coli, and characterize their structures and activities.

项目概要/摘要 黄素依赖性卤化酶 (FDH) 是来自细菌和真菌的生物合成酶 催化与各种芳香族底物形成区域选择性 C-X 键（X=Cl、Br、I）。 其区域选择性和环境友好的反应条件使 FDH 具有吸引力 用于过渡金属催化交叉反应的芳基卤化物绿色合成的候选者 偶联反应。拟议的项目将扩大我们对区域选择性芳基的理解 通过鉴定、结构和酶表征，FDH 形成卤化物 该酶家族的新成员，通过添加新成员来扩展 FDH 工具包 对目前表征的酶具有不同的底物和卤化物选择性。 在该项目的目标1中，我们将全面表征AbeH的扩展底物范围 和 BorH，我们已经证明来自土壤细菌宏基因组的两个 FDH 可以氯化和 溴化色氨酸产生两种不同的区域异构体。目标 2 和 3 基于我们 假设 FDH 也是由人类微生物组的细菌产生的，它们在那里 可能会产生在化学通讯中发挥作用的次生代谢物 细菌和人体细胞之间。这个假设得到了以下事实的支持：一个简单的 人类微生物组宏基因组霰弹枪三个序列文件的生物信息搜索 测序项目产生了 5 个 ORF，预计可编码新型 FDH。初步的 对五个假设的 FDH 基因中第一个的研究已证实它编码一种蛋白质 具有卤化吲哚的能力。目标 2 将涉及酶促反应的完整表征 这五个假定微生物组的活性、底物和卤化物范围以及晶体结构 外佣。目标 3 将建立生物信息学、酶学和结构学方面的培训计划 本科生物化学专业的生物学，谁将挖掘人类微生物组宏基因组 其他假定的 FDH 基因的序列，在大肠杆菌中表达和纯化基因产物， 并描述其结构和活动的特征。