Extending the biocatalytic scope of regiocomplementary flavin-dependent halogenase enzymes

Abstract

Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.

Fig. 1. The mechanism of the tryptophan 7-halogenase PrnA. (A) The flavin reductase from Escherichia coli (Fre) and glucose dehydrogenase (GDH2) from Bacillus megaterium were used to recycle FAD and NAD⁺. (B) PrnA active site with the tryptophan substrate bound. Residues which stack above and below the substrate indole are removed for clarity. The hypohalous acid generated (step i), reacts with the amino group of K79 (step ii) resulting in a chloroamine electrophile which attacks the indole C7 (step iii). E346 acts as a general base to deprotonate the σ-complex.

Fig. 2. Substrates and products from reactions with PyrH and PrnA.

Fig. 3. The % conversion of anthranilic 4, by PrnA mutants, and ratios of products 5- and 3-chloroanthranilic acid (4a & 4b) after 1 hour with halogenase enzyme (10 μM) and substrate (0.5 mM).

Fig. 4. X-ray crystal structure of PrnA F454K mutant (PDB 4Z44) with anthranilic acid (4) positioned in the active site showing possible H-bonding interactions with the substrate.