Tryptophan 7-halogenase (, PrnA, RebH) is an enzyme with systematic name L-tryptophan:FADH2 oxidoreductase (7-halogenating). This enzyme catalyses the following chemical reaction:
tryptophan + FADH2 + Cl− + O2 + H+ 7-chloro-L-tryptophan + FAD + 2 H2O
The enzyme can use bromide ions (Br−) in place of chloride (Cl−).
Background
Tryptophan 7-halogenase is a member of a class of enzymes known as flavin-dependent halogenases. Prior to tryptophan 7-halogenase's discovery, it was thought that all halogen atoms in metabolites were incorporated by the action of haloperoxidases, another class of halogenases dependent on metal centers such as vanadium or heme, or perhydrolases, a class of halogenases that generates peracids that in turn oxidize halide ions to hypohalous acids, which act as halogenating agents. These enzymes halogenate without substrate specificity and regioselectivity. The first tryptophan 7-halogenase was isolated in 1995 by Dairi et al. after a comparison in amino acid sequence revealed no similarity with previously known haloperoxidases. In contrast with haloperoxidases, flavin-dependent halogenases are much narrower in substrate scope and significantly more regioselective. Other flavin-dependent tryptophan halogenases include tryptophan 5-halogenase and tryptophan 6-halogenase.
Structure
Tryptophan 7-halogenase is a 538-residue, 61-kDa protein. In solution, a number of homologues exist as homodimers.
The enzyme has two main binding sites: one for FAD and one for tryptophan. Note that the FAD is supplied by a separate flavin reductase which may be a general enzyme recruited from metabolism or a specific enzyme encoded in the relevant biosynthetic gene cluster. The former site is also the site of reaction for the oxidation of FAD by O2. Connecting these two sites is a tunnel-like structure approximately 10 Å in length. The FAD binding site also binds a single chloride ion. A chlorinating agent is generated at the FAD-binding site, then channeled through this tunnel where it reacts with the substrate tryptophan. Tryptophan is bound by a number of interactions: other aromatic amino acid residues such as tryptophan, phenylalanine, and histidine seemingly "sandwich" the substrate, the N-H group of tryptophan forms hydrogen bonding interactions with the peptide backbone of the enzyme, and the amino acid moiety of tryptophan interacts with nearby tyrosine and glutamate residues. It is thought that the regioselectivity of the enzyme is a consequence of the spatial orientation of the tryptophan, with C7 placed close to the end of the tunnel where the chlorinating agent exits. Multiple halogenations are prevented as the addition of a halogen atom to the substrate greatly increases its steric bulk, causing it to dissociate from the enzyme.
Mechanism
Tryptophan 7-halogenases are FADH2-dependent, meaning they require an FADH2 cofactor in order to carry out their reaction. Flavin reductases are responsible for the conversion of FAD to FADH2. For sustained activity in an in vitro setting, tryptophan 7-halogenases thus require either excess FADH2 or the presence of a flavin reductase. Since flavin reductase is itself NAD(P)H-dependent, a recent work studying RebH used a cofactor regeneration system wherein glucose dehydrogenase reduces NAD(P)+ to NAD(P)H, which RebF (RebH's native flavin reductase partner) uses to reduce FAD to FADH2 for subsequent use in RebH’s active site. Mechanistically, the flavin-dependent halogenases are similar to flavin-containing monooxygenases. In the first step, FADH2 is oxidized by molecular oxygen, generating a high-energy flavin hydroperoxide.
At this point, there are two proposed mechanisms to achieve halogenation.
In the first, flavin hydroperoxide attacks the C6-C7 double bond of tryptophan, generating an epoxide. This epoxide is then opened by regioselective nucleophilic attack of halide anion at C7. The resultant halohydrin is dehydrated to form 7-chlorotryptophan. This mechanism is dubious due to the long channel separating the flavin cofactor from the tryptophan substrate.