Abstract
To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to
first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity
through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site
Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the co-factor-binding site is by direct reaction of molecular
oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the
halogenation active-site. Lys79, located in this site, has been proposed to direct HOCl towards Trp C7 through hydrogen bonding
or direct reaction with HOCl to form a -NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based
on MD simulations and active-site DFT ‘cluster’ models of FDH PrnA in complex with its native substrate L-tryptophan, hypochlorous
acid and FAD co-factor. MD simulations with different protonation states for key active-site residues suggest that Lys79
directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms.
first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity
through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site
Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the co-factor-binding site is by direct reaction of molecular
oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the
halogenation active-site. Lys79, located in this site, has been proposed to direct HOCl towards Trp C7 through hydrogen bonding
or direct reaction with HOCl to form a -NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based
on MD simulations and active-site DFT ‘cluster’ models of FDH PrnA in complex with its native substrate L-tryptophan, hypochlorous
acid and FAD co-factor. MD simulations with different protonation states for key active-site residues suggest that Lys79
directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms.
| Original language | English |
|---|---|
| Pages (from-to) | 15352–15360 |
| Journal | ACS Catalysis |
| Volume | 12 |
| Issue number | 24 |
| Early online date | 30 Nov 2022 |
| DOIs | |
| Publication status | Published - 16 Dec 2022 |