Vanadium-dependent bromoperoxidase (organic molecule bromination)

 

V-BPO, isolated from Ascophyllum nodosum, is a vanadium-dependent haloperoxidase.

It catalyses the oxidation of halides by hydrogen peroxide to form hypohalides, which can go on to react with organic substrates in halogenation reactions. In this entry, we show one of the side reactions of the vanadium-dependent haloperoxidases: bromination of phenols, the reaction produces 4-bromophenol in higher yields than the 2,4-dibromophenol. The enzyme is known to halogenate many other organic substrates including phenols, indoles, terpenes, and pyrroles.

V-BPO has the ability to oxidise bromide and, to a lesser extent, chloride and iodide.

V-BPO contains a vanadium(V) ion with a trigonal bipyramidal coordination sphere. It is coordinated to His486 and a hydroxide in the axial positions, and two oxygen atoms and a hydroxide in the equatorial positions.

 

Reference Protein and Structure

Sequence
P81701 UniProt (1.11.1.18) IPR016119 (Sequence Homologues) (PDB Homologues)
Biological species
Ascophyllum nodosum (Knotted wrack) Uniprot
PDB
1qi9 - X-RAY SIRAS STRUCTURE DETERMINATION OF A VANADIUM-DEPENDENT HALOPEROXIDASE FROM ASCOPHYLLUM NODOSUM AT 2.0 A RESOLUTION (2.05 Å) PDBe PDBsum 1qi9
Catalytic CATH Domains
1.10.606.10 CATHdb (see all for 1qi9)
Cofactors
Vanadate(3-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.11.1.18)

hydrogen peroxide
CHEBI:16240ChEBI
+
bromide
CHEBI:15858ChEBI
+
phenol
CHEBI:15882ChEBI
→
2,4-dibromophenol
CHEBI:34238ChEBI
+
water
CHEBI:15377ChEBI
+
hydron
CHEBI:15378ChEBI
+
4-bromophenol
CHEBI:47248ChEBI
Alternative enzyme names: Bromoperoxidase, Haloperoxidase, Eosinophil peroxidase,

Enzyme Mechanism

Introduction

An oxygen of hydrogen peroxide attacks the vanadium centre and causes the elimination of the axial hydroxide, which then deprotonates the bound peroxide to form water. The second oxygen of the peroxide ligand then attacks the vanadium centre and causes the elimination of an equatorial oxygen, which then abstracts the second peroxide proton. This forms a side-on vanadium-peroxo species. Bromide attacks one of the oxygens of the peroxo group, causing the O-O bond to break and leading to the formation of an axial vanadium-OBr complex.

The substrate hydrocarbon then binds in the active site and is bromonated by the activated O-Br species.

Catalytic Residues Roles

UniProt PDB* (1qi9)
Lys341 Lys341A Lys341 is thought to polarise the vanadium-bound peroxo group and increase its susceptibility to attack by bromide. Lys341 forms a hydrogen bond to His411 and this is thought to modulate its polarising power. This is thought to be partially responsible for the selectivity of the enzyme for bromide over chloride. electrostatic stabiliser
His411 His411A His411 forms a hydrogen bond to Lys341, buffering its polarising power towards the peroxo group. This is thought to be partially responsible for the selectivity of the enzyme for bromide over chloride. increase basicity, increase electrophilicity
His418 His418A His418 increases the nucleophilicity of the axial hydroxide, thus making it more able to abstract a proton from hydrogen peroxide. electrostatic stabiliser
His486 His486A His486 coordinates to the vanadate ion and is thought to be necessary for the formation of the vanadium-peroxo intermediate. It is thought that it is responsible for the lengthening and possible weakening of the axial V-O bond. activator, metal ligand
Asp490 Asp490A Helps activate and position the vanadate binding His486. activator
Arg349, Ser416, Arg480, Gly417 (main-N) Arg349A, Ser416A, Arg480A, Gly417A (main-N) Forms the positive binding site for the cofactor; activating and stabilising it. electrostatic stabiliser
*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

proton transfer, bimolecular nucleophilic substitution, dehydration, intramolecular nucleophilic substitution, bimolecular nucleophilic addition, aromatic bimolecular electrophilic addition, inferred reaction step, aromatic bimolecular elimination

References

  1. Wischang D et al. (2012), Tetrahedron, 68, 9456-9463. Bromination of phenols in bromoperoxidase-catalyzed oxidations. DOI:10.1016/j.tet.2012.08.081.
  2. Frank A et al. (2016), Chembiochem, 17, 2028-2032. Characterization of a Cyanobacterial Haloperoxidase and Evaluation of its Biocatalytic Halogenation Potential. DOI:10.1002/cbic.201600417. PMID:27542168.
  3. Rehder D (2015), Metallomics, 7, 730-742. The role of vanadium in biology. DOI:10.1039/c4mt00304g. PMID:25608665.
  4. Wischang D et al. (2013), Dalton Trans, 42, 11926-. Vanadate-dependent bromoperoxidases from Ascophyllum nodosum in the synthesis of brominated phenols and pyrroles. DOI:10.1039/c3dt51582f. PMID:23881071.
  5. Littlechild J et al. (2009), J Inorg Biochem, 103, 617-621. Vanadium containing bromoperoxidase – Insights into the enzymatic mechanism using X-ray crystallography. DOI:10.1016/j.jinorgbio.2009.01.011. PMID:19230976.
  6. Weyand M et al. (1999), J Mol Biol, 293, 595-611. X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 Å resolution. DOI:10.1006/jmbi.1999.3179. PMID:10543953.
  7. Butler A (1998), Curr Opin Chem Biol, 2, 279-285. Vanadium haloperoxidases. DOI:10.1016/s1367-5931(98)80070-7.
  8. Tschirret-Guth RA et al. (1994), J Am Chem Soc, 116, 411-412. Evidence for organic substrate binding to vanadium bromoperoxidase. DOI:10.1021/ja00080a063.

Step 1. The axial hydroxide of the vanadate cofactor deprotonates hydrogen peroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His486A metal ligand
Lys341A electrostatic stabiliser
Arg349A electrostatic stabiliser
His411A increase basicity
Ser416A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
His418A electrostatic stabiliser
Arg480A electrostatic stabiliser
Asp490A activator
His486A activator

Chemical Components

proton transfer

Step 2. The activated hydrogen peroxide initiates a nucleophilic attack on the vanadate in a substitution reaction, eliminating water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His486A activator
Asp490A activator
Arg480A electrostatic stabiliser
His418A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
Ser416A electrostatic stabiliser
Arg349A electrostatic stabiliser
Lys341A electrostatic stabiliser
His486A metal ligand
His411A increase electrophilicity

Chemical Components

ingold: bimolecular nucleophilic substitution, dehydration

Step 3. One of the equatorial oxo groups deprotonates the attached hydrogen peroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys341A electrostatic stabiliser
Arg349A electrostatic stabiliser
Ser416A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
His418A electrostatic stabiliser
Arg480A electrostatic stabiliser
Asp490A activator
His486A activator
His486A metal ligand

Chemical Components

proton transfer

Step 4. The peroxide initiates a nucleophilic attack on the vanadate in a substitution reaction, eliminating hydroxide and forming a three membered ring.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His486A activator
Asp490A activator
Arg480A electrostatic stabiliser
His418A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
Ser416A electrostatic stabiliser
Arg349A electrostatic stabiliser
Lys341A electrostatic stabiliser
His486A metal ligand

Chemical Components

ingold: intramolecular nucleophilic substitution

Step 5. The halide adds to one of the peroxide oxygen atoms.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys341A electrostatic stabiliser
Arg349A electrostatic stabiliser
Ser416A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
His418A electrostatic stabiliser
Arg480A electrostatic stabiliser
Asp490A activator
His486A activator
His486A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition

Step 6. The phenol attacks the electrophilic bromo species.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His486A activator
Asp490A activator
Arg480A electrostatic stabiliser
His418A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
Ser416A electrostatic stabiliser
Arg349A electrostatic stabiliser
Lys341A electrostatic stabiliser

Chemical Components

ingold: aromatic bimolecular electrophilic addition

Step 7. The highly active oxo species of the vanadate cofactor abstracts the proton from the intermediate, regenerating both the cofactor and the aromaticity of the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys341A electrostatic stabiliser
Arg349A electrostatic stabiliser
Ser416A electrostatic stabiliser
Gly417A (main-N) electrostatic stabiliser
His418A electrostatic stabiliser
Arg480A electrostatic stabiliser
Asp490A activator
His486A activator
His486A metal ligand

Chemical Components

inferred reaction step, proton transfer, ingold: aromatic bimolecular elimination
Download: Image, Marvin File

Step 1. The axial hydroxide of the vanadate cofactor deprotonates hydrogen peroxide.

Step 2. The activated hydrogen peroxide initiates a nucleophilic attack on the vanadate in a substitution reaction, eliminating water.

Step 3. One of the equatorial oxo groups deprotonates the attached hydrogen peroxide.

Step 4. The peroxide initiates a nucleophilic attack on the vanadate in a substitution reaction, eliminating hydroxide and forming a three membered ring.

Step 5. The halide adds to one of the peroxide oxygen atoms.

Step 6. The phenol attacks the electrophilic bromo species.

Step 7. The highly active oxo species of the vanadate cofactor abstracts the proton from the intermediate, regenerating both the cofactor and the aromaticity of the product.

Products.

Contributors

Gemma L. Holliday