Discovery and Characterization of Novel Halogenases from the Human Microbiome

项目概要/摘要 黄素依赖性卤代酶（flavin-dependent halogenases，FDH）是来源于细菌和真菌的生物合成酶 其催化与各种芳族底物区域选择性C-X键形成（X = Cl、Br、I）。 它们的区域选择性和环境友好的反应条件使FDH具有吸引力 用于过渡金属催化交叉偶联的芳基卤的绿色合成的候选物， 偶联反应该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， 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。 在本项目的目标1中，我们将充分表征AbeH的扩展底物范围 和BorH，两种来自土壤细菌宏基因组的FDH，我们已经证明它们可以氯化， 溴化色氨酸以产生两种不同的区域异构体。目标2和3基于我们的 假设FDH也是由人类微生物组的细菌产生的， 可能产生次级代谢物，在化学通讯中发挥作用， 细菌和人类细胞之间的关系。这一假设得到以下事实的支持： 人类微生物宏基因组鸟枪法三个序列文件的生物信息学搜索 测序项目产生了五个预测编码新型FDH的ORF。初步 对五个假设的FDH基因中的第一个的研究已经证实，它编码一种蛋白质， 具有卤化吲哚的能力。目标2将涉及酶的全面表征 活性、底物和卤化物范围以及这五种假定微生物组的晶体结构 外佣。目标3将建立一个生物信息学、酶学和结构 生物学本科生生物化学专业，谁将挖掘人类微生物组宏基因组 另外的推定FDH基因的序列，在E.大肠杆菌， 并描述它们的结构和活动。

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.