Discovery and Characterization of Novel Halogenases from the Human Microbiome and from Archaea

University Of Toledo, Toledo OH

Investigators

Linked publications, trials & patents

Abstract

PROJECT SUMMARY/ABSTRACT Flavin-dependent halogenases (FDHs) are biosynthetic enzymes from bacteria and fungi that catalyze regioselective C-X bond formation (X=Cl, Br) 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 Specific Aim 1 of this project, we will continue our characterization 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. In the last project period, we have identified 21 and 25 additional substrates that can be halogenated by BorH and AbeH have begun the process of identifying and characterizing the specific halogenated products, which include pharmaceutically relevant compounds such as 4-chlorokynurenine and 1-(4-chlorophenyl)-piperazine. Aim 1 will also investigate the full substrate scope of Hal4, a novel FDH produced by the human microbiome that we recently identified. We also intend to continue our crystallographic analysis of FDH- substrate interactions by solving crystal structures of BorH and Hal4 with various substrates bound to understand how the enzyme recognizes, binds, and orients these substrates to produce specific regioisomers. Specific Aim 2 continues the work that we began during the last project period with the identification of Hal4 and involves continued the data mining of human microbiome metagenomes for genes that encode novel FDHs. Our discovery of Hal4 validated our hypothesis that FDHs are produced by the bacteria of the human microbiome, where they may be involved in generate secondary metabolites that play a role in chemical communication between bacteria and human cells. We have identified an additional 25 putative FDH genes from the human microbiome as well as two from archaea and two from other bacterial metagenomes, and these will be expressed and purified so their structures and activities can be characterized. Specific Aim 3 is a new aim for this renewal and involves the investigation of differences in absorbance and fluorescence properties between various FDH substrates and their halogenated products for the purposes of identifying wavelengths and methods that can be applied to continuous real-time assays for FDH activity, for use in high-throughput screening of novel FDHs as well as for kinetic analysis.