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.

Genomic Determinants Encode the Reactivity and Regioselectivity of Flavin-Dependent Halogenases in Bacterial Genomes and Metagenomes

“…In this study, we found primarily genomic patterns that determine the enzymatic activity toward tryptophan (Trp-FDH versus non-Trp-FDH) and the site of halogenation for tryptophan (5-, 6-, and 7-Cl/Br-Trp-FDH). Recent studies found that the sequences with different substrate scope were clustered based on the sequence similarity in the network analysis ( 19 ) and screened the halogenation with various substrates ( 65 ). Structure analysis of the known proteins identified the residues that bind to FAD and substrates ( 66 – 69 ).…”

Two Novel, Flavin-Dependent Halogenases from the Bacterial Consortia of Botryococcus braunii Catalyze Mono- and Dibromination

“…A pharmacophore model was adapted from a virtual screening methodology for BrvH, and revealed 19 compounds halogenated by BrvH [52]. SpH1 is highly similar to BrvH and clusters within one clade with BrvH.…”

“…The activities of PrnF and ADH were determined as published by Frese et al [3]. The halogenation activities of SpH1 and SpH2 towards different substrates were carried out for 48 h at 25 • C and 500 rpm in a total volume of 0.5 mL, containing 1.25 mg/mL enzyme, 1 mM substrate, 1 µM FAD, 100 µM NAD, 100 mM NaBr/NaCl, RR-ADH (2 U/mL) PrnF (2.5 U/mL), 5% (v/v) iso-propanol, and 100 mM Na 2 HPO 4 buffer (pH 7.4) [33,52]. Samples were quenched by adding an equal volume of methanol.…”

“…One of them, BrvH, was characterized with respect to halide and substrate preference in vitro, leading to the conclusion that it halogenates indole and indole derivatives, as well as phenol derivatives, and prefers bromination over chlorination. BrvH was crystallized and revealed a structure similar to Trp halogenases, in addition to an open substrate binding site, which might lead to the acceptance of larger substrates, such as peptides [33,52]. BrvH was the first reported flavin-dependent halogenase that accepts chloride and bromide with a preference for bromination over chlorination.…”

Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds

“…To improve the utility of promiscuous enzymes, studies have implemented rational enzyme engineering, directed evolution and synthetic biology to assess and improve substrate scope (Glenn et al, 2011;Payne et al, 2015;Shepherd et al, 2015;Neubauer et al, 2020), regio-specificity (Lang et al, 2011;Andorfer et al, 2016;Shepherd et al, 2016;Moritzer et al, 2019), stability (Payne et al, 2013;Poor et al, 2014;Minges et al, 2020) and activity (Andorfer et al, 2016(Andorfer et al, , 2017Kong et al, 2020) of FDH halogenases (Table 3). Of particular interest are a study deploying directed evolution to yield RebH variants targeting positions 5, 6, and 7 of tryptamine (Andorfer et al, 2016), which could help to avoid the tryptophan decarboxylase bottleneck, and a study in which the substrate specificity of RebH was evolved to target, albeit with lower activities, "latestage" MIAs such as the yohimbines (Payne et al, 2015), which may allow promiscuity requirements to be eschewed entirely.…”