Table 1.
Program for the site-directed mutagenesis PCR.
Fig 1.
Phylogenetic tree of positive detected FHals from the Botany Bay metagenome in comparison to known FHals on amino acid level.
The tree was constructed using neighbour joining method, bootstrap 1000, with the alignment based on amino acids by MEGA7 [43] and MUSCLE [39]. 1–42: positive FHal hits from the Botany Bay metagenome; PyrH, PrnA, RebH, Thal: Tryptophan halogenases belonging to variant A and accept free substrate; PltA, CrpH, BhaA, CndH: FHals belonging to variant B and require carrier bound substrate. The amino acid sequences are shown in S3 Table.
Fig 2.
Gene cluster in the vicinity of gene 12 (K) in the Botany Bay metagenome.
Upstream to the gene 12 (K), another FHal, 13 (L) was identified.
Fig 3.
HPLC traces of the enzymatic halogenation of l-tryptophan (1 mM) and indole (1 mM) by BrvH (in cell lysate for 19 h in presence of 100 mM NaCl or NaBr).
l-tryptophan (tR: 2.2 minutes) is not being halogenated, while Indole (tR: 3.2 minutes) is fully converted within 19 h to bromoindole (tR: 3.8 minutes) (HPLC method A).
Fig 4.
Time course of the conversion of 0.05 mM indole by BrvH with NaBr over 25 minutes at 25 °C.
The conversion rate was identified via RP-HPLC by determining the ratio of the peak areas of indole and bromoindole. The specific activity was defined between 0 and 15 minutes reaction time.
Fig 5.
HPLC traces of enzyme products of BrvH incubated with indole and NaBr/NaCl and the same products spiked with commercially available 3-chloroindole.
BrvH incubated with indole and NaBr leads to bromoindole (tR: 5.2 min) and incubated with NaCl leads to chloroindole (tR: 4.6 min). Both enzyme products spiked with 3-chloroindole (tR: 4.6 min) show that the halogenated product of BrvH incubated with indole and NaCl has the same retention time with commercially 3-chloroindole (HPLC method D).
Fig 6.
HPLC traces of the halogenation of indole with BalH in excess of NaCl (100 mM) and three different concentrations of NaBr (0 mM, 10 mM, 100 mM).
Even in 10 times higher concentrations of chloride over bromide, bromide is the preferred halide source (HPLC method A).
Fig 7.
Structure of apo BrvH compared to RebH bound to FAD, Trp and Cl-.
A: The structural alignment was made in PyMOL based on the conserved “box” domain of the two halogenases (BrvH: 6–99, 162–416, 494–502 vs. RebH: 2–98, 167–426, 519–528; PDB ID 2oa1). FAD, Trp (both dark grey) and Cl- (green) are present only in the structure of RebH. BrvH (blue) and RebH (grey) are structurally very similar. A major difference is found in the substrate binding site, which is accessible to the solvent in BrvH, while it is covered by a longer loop in RebH. B: Empty substrate binding site of BrvH overlaid with the tryptophan binding site of RebH containing Trp (dark grey). Several of the RebH residues directly contacting the bound Trp are shown as grey sticks along with the corresponding residues of BrvH shown in blue. The loops that are shown correspond to those in A. While the residues contacting the indole ring are conserved, those residues that form hydrogen bonds to the carboxylate and the α-amino group of Trp in RebH are not present in BrvH. K83 and E349 of BrvH correspond to the amino acids shown to be important for the catalytic mechanism in other halogenases (K79 and E357 in RebH).
Fig 8.
Comparison of the halide binding site in RebH and BrvH. FAD (dark grey) and Cl- (green) are present only in the structure of RebH.
Coordination of the halide takes place via the amide nitrogens of the backbone. Whilst the halide binding motif in RebH (grey) is T359, G360 in BrvH (blue) we see a T351, S352 at the same site. However, the amino acid exchange does not seem to alter the overall structure of the halide binding site, thus not giving a definite hint as to why BrvH would preferentially brominate.