Abstract
Flavin-dependent halogenases catalyse halogenation of aromatic compounds. In most cases, this reaction proceeds with high regioselectivity and requires only the presence of FADH2, oxygen, and halide salts. Since marine habitats contain high concentrations of halides, organisms populating the oceans might be valuable sources of yet undiscovered halogenases. A new Hidden-Markov-Model (HMM) based on the PFAM tryptophan halogenase model was used for the analysis of marine metagenomes. Eleven metagenomes were screened leading to the identification of 254 complete or partial putative flavin-dependent halogenase genes. One predicted halogenase gene (brvH) was selected, codon optimised for E. coli, and overexpressed. Substrate screening revealed that this enzyme represents an active flavin-dependent halogenase able to convert indole to 3-bromoindole. Remarkably, bromination prevails also in a large excess of chloride. The BrvH crystal structure is very similar to that of tryptophan halogenases but reveals a substrate binding site that is open to the solvent instead of being covered by a loop.
Highlights
In many bioactive compounds, halogen substituents are an important factor for the biological activity [1]
The fact that all predicted genes cluster closer to the tryptophan halogenases may be caused by the selection criteria since our HMM consists of the PFAM model of tryptophan halogenases
The e-value and the gene size resembling the length of known tryptophan halogenases were used as criteria for selection
Summary
Halogen substituents are an important factor for the biological activity [1]. Haloperoxidases and cofactor- and metal-free haloperoxidases (perhydrolases) were the first known halogenating enzymes. These require hydrogen peroxide for the halogenation reaction and lack substrate specifity and regioselectivity [2,3]. Flavin-dependent halogenases (FHals) play an essential role in the regioselective halogenation of natural products such as chloramphenicol [4,5], vancomycin [6,7], or cryptophycin [8,9]. Chemical halogenation of aromatic compounds often requires harsh reaction conditions using catalysts like Lewis acids and often lacks regioselectivity. FHals have the potential to overcome the drawbacks of conventional chemical approaches, as they work under more environmentally friendly conditions [10,11]. Oxygen, and the cofactor FADH2 are required
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