Further Characterization of Fungal Halogenase RadH and Its Homologs

Abstract

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.

Keywords: X-ray crystallography; flavin-dependent halogenases; halogenation; heteroaromatic.

Figure 1

Activity of RadH and the two active homologs against substrate, 6-hydroxyisoquinoline. Conditions: 6-OH isoquinoline (1.0 mM), RadH Biocatalyst (2.0 mol%), FAD (1.0 mol%), Fre (0.4 mol%), NADH (5.0 equiv), MgCl₂ (10.0 equiv), Tris HCl buffer (10 mM, pH 7.4), 18 h. Control reaction conditions: 6-OH isoquinoline (1.0 mM), FAD (1.0 mol%), Fre (0.4 mol%), NADH (5.0 equiv), MgCl₂ (10.0 equiv), Tris HCl buffer (10 mM, pH 7.4), and 18 h. Conversion (%) represents the area under the peak (at 254 nm UV absorbance) for the desired chlorinated product (Cl-product) relative to the total area of the Cl-product and remaining starting substrate.

Figure 2

Substrate specificities profile of RadH and the two homologs against a panel of 15 substrates under the halogenation assay. Conditions: substrate (0.5 mM), RadH (3.0 mol%), FAD (0.2 mol%), Fre (0.5 mol%), NADH (5.0 equiv), Glucose (10.0 equiv), GdHi (0.5 mol%), MgCl₂ (20.0 equiv), and phosphate buffer (10 mM, pH 7.4). Estimated relative % conversions (based on area under the LCMS peaks at 254 nm of starting material and respective product) were color-coded: light green (<5%) and dark green (x ≥ 5%). Grey boxes = not detected by mass spectroscopy.

Figure 3

Specificities of RadH. Conditions: Substrate (0.5 mM), RadH Biocatalyst (3 mol%), GdHi (0.5 mol%), FAD (0.2 mol%), Fre (0.5 mol%), NADH (5.0 equiv), Glucose (10.0 equiv), MgCl₂ (20.0 equiv), and phosphate buffer (10 mM, pH 7.4). Conversions are determined as an average of at least two runs by comparison of the LC peak areas between crude reaction mixtures relative to the control standard. A standard control experiment refers to the reaction setup without the addition of RadH. N.D. = not detected.

Figure 4

(a) Crystal structure of RadH (PDB: 8GU0) with FAD and chlorine atom bound. Main structural features are colored accordingly: GxGxxG domain (blue), Fx.Px.Sx.G domain (yellow), WxWxIP domain (orange), and catalytic lysine residue [8,26] (pink). The additional loop region of RadH (purple) and C-terminus region (red) of RadH are structurally different from other halogenases. (b) A zoom-in view of active site residues of RadH. The highest confidence docking pose of 6-hydroxyquinoline in the active site of RadH shown as spheres. (c) Overlay of the C-terminus of RadH (red) over the electrostatic potential map of CndH (PDB: 3E1T). (d) Overlay of the C-terminus of PrnA (PDB: 2AR8) (yellow) over the electrostatic potential map of CndH. The tyrosine substrate bound to PrnA is sitting at the proposed active site of CndH.