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.

项目摘要/摘要 黄素依赖性卤代酶（FDHS）是来自细菌和真菌的生物合成酶 与各种芳族底物相结合的C-X键形成（X = Cl，Br，I）。 他们的区域选择性和环境友好的反应条件使FDH具有吸引力 用于用于过渡金属催化的交叉的芳基卤化物绿色合成的候选者 耦合反应。拟议的项目将扩大我们对区域选择性芳基的理解 FDHS通过识别以及结构和酶促表征形成卤化物 这个酶家族的新颖成员，通过添加新成员来扩展FDH工具包 具有不同的底物和卤化物的选择性，目前具有特征性的酶。 在该项目的AIM 1中，我们将充分表征ABEH的扩展基板范围 和Borh，我们显示的两种来自土壤细菌元基因组的FDH可以氯可以氯化和 溴化色氨酸产生两个不同的区域异构体。目标2和3是基于我们的 假设FDH也由人类微生物组的细菌产生 可能会产生在化学通信中发挥作用的次级代谢产物 在细菌和人类细胞之间。一个简单的事实证明了这一假设 从人类微生物组元基因组shot弹枪中搜索三个序列文件的生物信息学搜索 测序项目产生了预测的五个ORF编码新颖的FDH。初步的 对五个假设的FDH基因中第一个的研究证实了它编码蛋白质 具有卤代吲哚的能力。 AIM 2将涉及酶促的全部表征 这五个推定的微生物组的活性，底物和卤化物范围以及晶体结构 FDHS。 AIM 3将建立生物信息学，酶学和结构的培训计划 本科生物化学专业的生物学，他们将挖掘人类微生物组元基因组 其他推定FDH基因的序列，在大肠杆菌中表达和净化基因产物， 并描述其结构和活动。