Dataset Information

Predicting the Substrate Scope of the Flavin-Dependent Halogenase BrvH.

ABSTRACT: The recently described flavin-dependent halogenase BrvH is able to catalyse both the bromination and chlorination of indole, but shows significantly higher bromination activity. BrvH was annotated as a tryptophan halogenase, but does not accept tryptophan as a substrate. Its native substrate remains unknown. A predictive model with the data available for BrvH was analysed. A training set of compounds tested in?vitro was docked into the active site of a complete protein model based on the X-ray structure of BrvH. The atoms not resolved experimentally were modelled by using molecular mechanics force fields to obtain this protein model. Furthermore, docking poses for the substrates and known non-substrates have been calculated. Parameters like distance, partial charge and hybridization state were analysed to derive rules for predicting activity. With this model for activity of the BrvH, a virtual screening suggested several structures for potential substrates. Some of the compounds preselected in this way were tested in?vitro, and several could be verified as convertible substrates. Based on information on halogenated natural products, a new dataset was created to specifically search for natural products as substrates/products, and virtual screening in this database yielded further hits.

SUBMITTER: Neubauer PR

PROVIDER: S-EPMC7754283 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Predicting the Substrate Scope of the Flavin-Dependent Halogenase BrvH.

Neubauer Pia R PR Pienkny Silke S Wessjohann Ludger L Brandt Wolfgang W Sewald Norbert N

Chembiochem : a European journal of chemical biology 20200804 22

The recently described flavin-dependent halogenase BrvH is able to catalyse both the bromination and chlorination of indole, but shows significantly higher bromination activity. BrvH was annotated as a tryptophan halogenase, but does not accept tryptophan as a substrate. Its native substrate remains unknown. A predictive model with the data available for BrvH was analysed. A training set of compounds tested in vitro was docked into the active site of a complete protein model based on the X-ray s ...[more]

PMID: 32645255

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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.

Dataset Information

Predicting the Substrate Scope of the Flavin-Dependent Halogenase BrvH.

Publications

Predicting the Substrate Scope of the Flavin-Dependent Halogenase BrvH.

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