Tryptophan 7-halogenase

 

Tryptophan 7-halogenase catalyses the chlorination of free tryptophan to 7-chlorotryptophan, which is the first step in the synthesis of the antibiotic pyrrolnitrin. Enzyme-catalysed chlorination of organic compounds in the laboratory remains a challenge, thus understanding the reaction mechanism of tryptophan 7-halogenase is important.

 

Reference Protein and Structure

Sequence
P95480 UniProt (1.14.19.9) IPR033856 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas fluorescens (Bacteria) Uniprot
PDB
2ar8 - The structure of tryptophan 7-halogenase (PrnA)suggests a mechanism for regioselective chlorination (2.2 Å) PDBe PDBsum 2ar8
Catalytic CATH Domains
3.50.50.60 CATHdb (see all for 2ar8)
Cofactors
Nadp zwitterion (1)
Click To Show Structure

Enzyme Reaction (EC:1.14.19.9)

chloride
CHEBI:17996ChEBI
+
FADH2(2-)
CHEBI:58307ChEBI
+
L-tryptophan zwitterion
CHEBI:57912ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
7-chloro-L-tryptophan zwitterion
CHEBI:58713ChEBI
+
FAD(3-)
CHEBI:57692ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: PrnA, RebH, PrnA (gene name), RebH (gene name), KtzQ (gene name),

Enzyme Mechanism

Introduction

In the FAD binding site, FADH2 reacts with molecular oxygen and subsequently with chlorine to form hypochlorous acid (HOCl). HOCl moves through a tunnel in the enzyme and becomes activated (Lys79) in order to chlorinate the substrate in the tryptophan binding site. The chlorine atom is inserted forming an arenium ion (Wheland intermediate). Proton removal of this reactive intermediate by Glu346 leads to the formation of the 7-chlorotryptophan product. The mechanism is regioselective. An alternative is the electrophilic attack performed by a preliminarily formed Lys79-bound chloramine. However, chloramines are milder chlorinating agents than HOCl and not sufficient for the reaction.

Catalytic Residues Roles

UniProt PDB* (2ar8)
Lys79 Lys79A Activates HOCl for electrophilic substitution on the tryptophan substrate. activator, hydrogen bond donor, proton donor
Glu346 Glu346A Deprotonates Wheland intermediate forming the product. proton acceptor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

inferred reaction step, hydride transfer, cofactor used, electron transfer, proton transfer, radical formation, intermediate formation, colligation, radical termination, bimolecular nucleophilic substitution, dehydration, aromatic bimolecular electrophilic addition, aromatic bimolecular elimination, overall product formed, rate-determining step

References

  1. Karabencheva-Christova TG et al. (2017), Sci Rep, 7, 17395-. Mechanistic Insights into the Reaction of Chlorination of Tryptophan Catalyzed by Tryptophan 7-Halogenase. DOI:10.1038/s41598-017-17789-x. PMID:29234124.
  2. Romero E et al. (2018), Chem Rev, 118, 1742-1769. Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes. DOI:10.1021/acs.chemrev.7b00650. PMID:29323892.
  3. Agarwal V et al. (2017), Chem Rev, 117, 5619-5674. Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse. DOI:10.1021/acs.chemrev.6b00571. PMID:28106994.

Step 1. NADPH donates a hydride to FAD. Mechanism inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

inferred reaction step, hydride transfer, cofactor used

Step 2. Proton-coupled electron transfer (PCET) from FADH- to molecular oxygen and to the C4a-carbon atom of the isoalloxazine ring, leading to formation of FADH• and OOH• radicals. Inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

electron transfer, proton transfer, radical formation, cofactor used, intermediate formation

Step 3. FADH• and OOH• radicals colligate, in which each of the molecular fragments donate one of the bonding electrons, resulting in formation of C4a-hydroperoxyflavin (C4aOOH) intermediate. Inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

colligation, inferred reaction step, radical termination

Step 4. A chloride anion makes a nucleophilic attack on the flavin peroxide yielding hypochlorous acid (HClO) and C(4a) hydroxyflavin.

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Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, intermediate formation, cofactor used

Step 5. Dehydration of C(4a) hydroxyflavin gives oxidised FAD (not shown). HOCl formed in the FAD binding site moves through a tunnel in the enzyme and becomes activated to chlorinate the substrate in the tryptophan binding site.

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Catalytic Residues Roles

Residue Roles

Chemical Components

dehydration

Step 6. A hydrogen bonding interaction between HOCl and Lys79 activates HOCl by increasing its electrophilicity. The chlorine atom is inserted and forms a Wheland intermediate.

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Catalytic Residues Roles

Residue Roles
Lys79A activator, hydrogen bond donor, proton donor

Chemical Components

ingold: aromatic bimolecular electrophilic addition, proton transfer

Step 7. Deprotonation of the Wheland intermediate by Glu346 forms the product. This is rate-limiting.

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Catalytic Residues Roles

Residue Roles
Glu346A proton acceptor

Chemical Components

ingold: aromatic bimolecular elimination, overall product formed, rate-determining step
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Step 1. NADPH donates a hydride to FAD. Mechanism inferred by curator.

Step 2. Proton-coupled electron transfer (PCET) from FADH- to molecular oxygen and to the C4a-carbon atom of the isoalloxazine ring, leading to formation of FADH• and OOH• radicals. Inferred by curator.

Step 3. FADH• and OOH• radicals colligate, in which each of the molecular fragments donate one of the bonding electrons, resulting in formation of C4a-hydroperoxyflavin (C4aOOH) intermediate. Inferred by curator.

Step 4. A chloride anion makes a nucleophilic attack on the flavin peroxide yielding hypochlorous acid (HClO) and C(4a) hydroxyflavin.

Step 5. Dehydration of C(4a) hydroxyflavin gives oxidised FAD (not shown). HOCl formed in the FAD binding site moves through a tunnel in the enzyme and becomes activated to chlorinate the substrate in the tryptophan binding site.

Step 6. A hydrogen bonding interaction between HOCl and Lys79 activates HOCl by increasing its electrophilicity. The chlorine atom is inserted and forms a Wheland intermediate.

Step 7. Deprotonation of the Wheland intermediate by Glu346 forms the product. This is rate-limiting.

Products.

Contributors

Noa Marson, Marko Babić