Discovery and Characterization of Novel Halogenases from the Human Microbiome

项目摘要/摘要 黄素依赖卤代酶(FDHS)是细菌和真菌的生物合成酶 它催化与各种芳香族底物形成区域选择性的C-X键(X=氯、溴、碘)。 他们的地区选择性和环境友好的反应条件使外籍家庭佣工具有吸引力 过渡金属催化的卤代芳烃的绿色合成候选化合物 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 外佣形成卤化物的鉴定及结构和酶特性研究 这个酶家族的新成员，通过添加新成员来扩展FDH工具包 对当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，我们将充分描述AbeH的扩展底物范围 和BorH，两个来自土壤细菌元基因组的外佣，我们已经证明了它们可以氯化和 将色氨酸溴化以产生两种不同的区域异构体。目标2和目标3基于我们的 假设外佣也是由人类微生物群中的细菌产生的，在那里他们 可能会产生在化学交流中起作用的次生代谢物 细菌和人类细胞之间的关系。这一假设得到了一个事实的支持，即一个简单的 人微生物组元基因组猎枪中三个序列文件的生物信息学搜索 测序项目产生了5个预测编码新的外佣的ORF。初步 对五个假想的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 这五个推定微生物组的活性、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 生物化学专业本科生生物学，谁将挖掘人类微生物组元基因组 其他可能的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.