Dataset Information

Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling.

ABSTRACT: Enzymes are powerful catalysts for site-selective C-H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence-function information on flavin-dependent halogenases (FDHs). This broad survey provided a number of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-molecule substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds were characterized and used for preparative-scale late-stage C-H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution.

SUBMITTER: Fisher BF

PROVIDER: S-EPMC6891866 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling.

Fisher Brian F BF Snodgrass Harrison M HM Jones Krysten A KA Andorfer Mary C MC Lewis Jared C JC

ACS central science 20191024 11

Enzymes are powerful catalysts for site-selective C-H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence-function information on flavin-dependent halogenases (FDHs). This broad survey provided a number of insights into FDH activity, including halide spec ...[more]

PMID: 31807686

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Project description:Xantholipin (compound 1), a polycyclic xanthone antibiotic, exhibited strong antibacterial activities and showed potent cytotoxicity. The biosynthetic gene cluster of compound 1 has been identified in our previous work, and the construction of xanthone nucleus has been well demonstrated. However, limited information of the halogenation involved in compound 1 biosynthesis is available. In this study, based on the genetic manipulation and biochemical assay, we characterized XanH as an indispensable flavin adenine dinucleotide (FAD)-dependent halogenase (FDH) for the biosynthesis of compound 1. XanH was found to be a bifunctional protein capable of flavin reduction and chlorination and exclusively used the NADH. However, the reduced flavin could not be fully and effectively utilized, and the presence of an extra flavin reductase (FDR) and chemical-reducing agent could promote the halogenation. XanH accepted its natural free-standing substrate with angular fused polycyclic aromatic systems. Meanwhile, it exhibited moderate halogenation activity and possessed high substrate specificity. The requirement of extra FDR for higher halogenation activity is tedious for future engineering. To facilitate efforts in engineering XanH derivative proteins, we constructed the self-sufficient FDR-XanH fusion proteins. The fusion protein E1 with comparable activities to that of XanH could be used as a good alternative for future protein engineering. Taken together, these findings reported here not only improve the understanding of polycyclic xanthones biosynthesis but also expand the substrate scope of FDH and pave the way for future engineering of biocatalysts for new active substance synthesis.IMPORTANCE Halogenation is important in medicinal chemistry and plays an essential role in the biosynthesis of active secondary metabolites. Halogenases have evolved to catalyze reactions with high efficiency and selectivity, and engineering efforts have been made to engage the selective reactivity in natural product biosynthesis. The enzymatic halogenations are an environmentally friendly approach with high regio- and stereoselectivity, which make it a potential complement to organic synthesis. FDHs constitute one of the most extensively elucidated class of halogenases; however, the inventory awaits to be expanded for biotechnology applications and for the generation of halogenated natural product analogues. In this study, XanH was found to reduce flavin and halogenated the freely diffusing natural substrate with an angular fused hexacyclic scaffold, findings which were different from those for the exclusively studied FDHs. Moreover, the FDR-XanH fusion protein E1 with comparable reactivity to that of XanH serves as a successful example of genetic fusions and sets an important stage for future protein engineering.

Project description:Halogenases create diverse natural products by utilizing halide ions and are of great interest in the synthesis of potential pharmaceuticals and agrochemicals. An increasing number of halogenases discovered in microorganisms are annotated as flavin-dependent halogenases (FDHs), but their chemical reactivities are markedly different and the genomic contents associated with such functional distinction have not been revealed yet. Even though the reactivity and regioselectivity of FDHs are essential in the halogenation activity, these FDHs are annotated inaccurately in the protein sequence repositories without characterizing their functional activities. We carried out a comprehensive sequence analysis and biochemical characterization of FDHs. Using a probabilistic model that we built in this study, FDHs were discovered from 2,787 bacterial genomes and 17 sediment metagenomes. We analyzed the essential genomic determinants that are responsible for substrate binding and subsequent reactions: four flavin adenine dinucleotide-binding, one halide-binding, and four tryptophan-binding sites. Compared with previous studies, our study utilizes large-scale genomic information to propose a comprehensive set of sequence motifs that are related to the active sites and regioselectivity. We reveal that the genomic patterns and phylogenetic locations of the FDHs determine the enzymatic reactivities, which was experimentally validated in terms of the substrate scope and regioselectivity. A large portion of publicly available FDHs needs to be reevaluated to designate their correct functions. Our genomic models establish comprehensive links among genotypic information, reactivity, and regioselectivity of FDHs, thereby laying an important foundation for future discovery and classification of novel FDHs. IMPORTANCE Halogenases are playing an important role as tailoring enzymes in biosynthetic pathways. Flavin-dependent tryptophan halogenases (Trp-FDHs) are among the enzymes that have broad substrate scope and high selectivity. From bacterial genomes and metagenomes, we found highly diverse halogenase sequences by using a well-trained profile hidden Markov model built from the experimentally validated halogenases. The characterization of genotype, steady-state activity, substrate scope, and regioselectivity has established comprehensive links between the information encoded in the genomic sequence and reactivity of FDHs reported here. By constructing models for accurate and detailed sequence markers, our work should guide future discovery and classification of novel FDHs.

Dataset Information

Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling.

Publications

Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling.

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