4-chlorobenzoyl-CoA dehalogenase

 

Chlorobenzoate dehalogenase catalyses the hydrolysis of chlorobenzoyl-CoA to hydroxybenzoyl-CoA. This reaction is used by bacteria as part of a three enzyme pathway for the utilisation of chlorinated organic compounds as a carbons source. The chlorobenzoate dehalogenase step is the second in the pathway and is structurally related to the crotonase-like superfamily of enzymes found in the beta-oxidation cycle.

Since chlorinated organic compounds are thought to have only been present in the biosphere in significant amounts for the latter half of this century due to industrial production. The potential for bacteria to evolve new degradation pathways within decades of exposure to a new compound offers the possibility of bioremediation of environmentally hazardous and toxic substances.

 

Reference Protein and Structure

Sequence
A5JTM5 UniProt (3.8.1.7) IPR001753 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas sp. CBS3 (Bacteria) Uniprot
PDB
1nzy - 4-CHLOROBENZOYL COENZYME A DEHALOGENASE FROM PSEUDOMONAS SP. STRAIN CBS-3 (1.8 Å) PDBe PDBsum 1nzy
Catalytic CATH Domains
3.90.226.10 CATHdb (see all for 1nzy)
Click To Show Structure

Enzyme Reaction (EC:3.8.1.7)

water
CHEBI:15377ChEBI
+
4-chlorobenzoyl-CoA
CHEBI:15498ChEBI
→
4-hydroxybenzoyl-CoA
CHEBI:15500ChEBI
+
hydrogen chloride
CHEBI:17883ChEBI

Enzyme Mechanism

Introduction

Aspartate 145 attacks the chlorobenzoate ring at the chlorinated carbon forming an arylated enzyme intermediate (Meisenheimer intermediate). The chloride then leaves and the enzyme returns to the resting state by attack of an activated water on the acyl carbon. Histidine 90 is thought to be the general base which activates the water based on mutagenic studies. The backbone amides of phenylalanine 64 and glycine 114 form an oxyanion hole for the stabilisation of the tetrahedral intermediate formed in the hydrolytic step [PMID:8679561].

Catalytic Residues Roles

UniProt PDB* (1nzy)
Ala86 (main-C) Ala86A (main-C) The main chain carbonyl activates the His90 for its general acid/base function. activator, hydrogen bond acceptor
His90 His90A General acid/base that abstracts a proton from Asp145, and later by the nucleophilic water. It is returned to its initial protonation state both times by the leaving group. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Gly114 (main-N), Phe64 (main-N) Gly114A (main-N), Phe64A (main-N) Back bond amide forms the oxyanion hole. hydrogen bond donor, electrostatic stabiliser
Trp137 Trp137A Stabilises the reaction intermediates. hydrogen bond donor, electrostatic stabiliser
Asp145 Asp145A Acts as the catalytic nucleophile. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, electrofuge, electrophile
*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

aromatic bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, aromatic unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, intermediate collapse, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, native state of enzyme regenerated, intermediate terminated

References

  1. Benning MM et al. (1996), Biochemistry, 35, 8103-8109. Structure of 4-Chlorobenzoyl Coenzyme A Dehalogenase Determined to 1.8 Å Resolution:  An Enzyme Catalyst Generated via Adaptive Mutation†,‡. DOI:10.1021/bi960768p. PMID:8679561.
  2. Xie D et al. (2005), J Phys Chem B, 109, 5259-5266. Theoretical Study of General Base-Catalyzed Hydrolysis of Aryl Esters and Implications for Enzymatic Reactions. DOI:10.1021/jp0506181. PMID:16863192.
  3. Xu D et al. (2005), FEBS Lett, 579, 4249-4253. Electrostatic influence of active-site waters on the nucleophilic aromatic substitution catalyzed by 4-chlorobenzoyl-CoA dehalogenase. DOI:10.1016/j.febslet.2005.06.056. PMID:16051230.
  4. Xu D et al. (2004), Chem Commun (Camb), 892-. A QM/MM study of a nucleophilic aromatic substitution reaction catalyzed by 4-chlorobenzoyl-CoA dehalogenase. DOI:10.1039/b401159g. PMID:15045116.
  5. Zheng Y et al. (1997), J Am Chem Soc, 119, 3868-3877. On the Dehalogenation Mechanism of 4-Chlorobenzoyl CoA by 4-Chlorobenzoyl CoA Dehalogenase:  Insights from Study Based on the Nonenzymatic Reaction. DOI:10.1021/ja970114j.
  6. Clarkson J et al. (1997), Biochemistry, 36, 10192-10199. Raman Study of the Polarizing Forces Promoting Catalysis in 4-Chlorobenzoate-CoA Dehalogenase†. DOI:10.1021/bi970941x. PMID:9254617.
  7. Yang G et al. (1996), Biochemistry, 35, 10879-10885. Identification of Active Site Residues Essential to 4-Chlorobenzoyl−Coenzyme A Dehalogenase Catalysis by Chemical Modification and Site Directed Mutagenesis†. DOI:10.1021/bi9609533. PMID:8718880.

Step 1. His90 deprotonates Asp145, which initiates a nucleophilic attack on the C4 of the 4-chlorobenzoyl-coenzyme A in an addition reaction. The conjugated double bonds rearrange to form an oxyanion, which is stabilised by the main chain amides of Phe64 and Gly114.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly114A (main-N) hydrogen bond donor
Trp137A hydrogen bond donor
Ala86A (main-C) hydrogen bond acceptor, activator
Asp145A hydrogen bond donor, hydrogen bond acceptor
His90A hydrogen bond acceptor, hydrogen bond donor
Phe64A (main-N) hydrogen bond donor
Asp145A proton donor
His90A proton acceptor
Asp145A nucleophile

Chemical Components

ingold: aromatic bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer

Step 2. The oxyanion collapses with rearrangement of the conjugated double bonds, eliminating chlorine, which deprotonates the His90.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly114A (main-N) hydrogen bond donor, electrostatic stabiliser
Trp137A hydrogen bond donor
Ala86A (main-C) hydrogen bond acceptor
Asp145A covalently attached, hydrogen bond acceptor
His90A hydrogen bond donor
Phe64A (main-N) hydrogen bond donor, electrostatic stabiliser
His90A proton donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, proton transfer

Step 3. His90 deprotonates water, which initiates a nucleophilic attack on the carboxylic carbon of the covalently attached Asp145, forming a new oxyanion, which is stabilised by Trp137.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly114A (main-N) hydrogen bond donor
Trp137A hydrogen bond donor
Ala86A (main-C) hydrogen bond acceptor, activator
Asp145A covalently attached, hydrogen bond acceptor
His90A hydrogen bond acceptor, hydrogen bond donor
Phe64A (main-N) hydrogen bond donor
His90A proton acceptor
Asp145A electrophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, proton transfer

Step 4. The oxyanion collapses, eliminating Asp145 as an electrofuge. The newly formed phenolic oxygen deprotonates His90.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly114A (main-N) hydrogen bond donor
Trp137A hydrogen bond donor, electrostatic stabiliser
Ala86A (main-C) hydrogen bond acceptor
Asp145A hydrogen bond acceptor
His90A hydrogen bond acceptor, hydrogen bond donor
Phe64A (main-N) hydrogen bond donor
His90A proton donor
Asp145A electrofuge, proton acceptor

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, native state of enzyme regenerated, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, proton transfer
Download: Image, Marvin File

Step 1. His90 deprotonates Asp145, which initiates a nucleophilic attack on the C4 of the 4-chlorobenzoyl-coenzyme A in an addition reaction. The conjugated double bonds rearrange to form an oxyanion, which is stabilised by the main chain amides of Phe64 and Gly114.

Step 2. The oxyanion collapses with rearrangement of the conjugated double bonds, eliminating chlorine, which deprotonates the His90.

Step 3. His90 deprotonates water, which initiates a nucleophilic attack on the carboxylic carbon of the covalently attached Asp145, forming a new oxyanion, which is stabilised by Trp137.

Step 4. The oxyanion collapses, eliminating Asp145 as an electrofuge. The newly formed phenolic oxygen deprotonates His90.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Alex Gutteridge, Craig Porter, Katherine Ferris