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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结/文摘