Limonene-1,2-epoxide hydrolase

 

Rhodococcus erythropolis is a species of bacteria able to grow on limonene. Its enzyme limonene epoxide hydrolase (LEH) is able to catalyse a key step in this process, namely the hydrolysis of limonene-1-2-epoxide to limonene-1-2-diol. However, the enzyme has a different fold and mechanism to the homologous family of epoxide hydrolases already known to exist, thus represents an example of convergent evolution. In fact, the enzyme bears little structural homology to any known enzyme except for kesosteriod isomerase, which catalyses a completely different reaction. The hydrolysis catalysed by LEH is enantioconvergent, therefore attracting wide interest in industrial applications.

 

Reference Protein and Structure

Sequence
Q9ZAG3 UniProt (3.3.2.8) IPR013100 (Sequence Homologues) (PDB Homologues)
Biological species
Rhodococcus erythropolis (Bacteria) Uniprot
PDB
1nww - Limonene-1,2-epoxide hydrolase (1.2 Å) PDBe PDBsum 1nww
Catalytic CATH Domains
3.10.450.50 CATHdb (see all for 1nww)
Click To Show Structure

Enzyme Reaction (EC:3.3.2.8)

(4R)-limonene 1,2-epoxide
CHEBI:35672ChEBI
+
water
CHEBI:15377ChEBI
→
(1S,2S,4R)-limonene-1,2-diol
CHEBI:18515ChEBI
Alternative enzyme names: Limonene oxide hydrolase,

Enzyme Mechanism

Introduction

The mechanism for the reaction proceeds in a single concerted step with concomitant nucleophilic attack by a water molecule and protonation of the epoxide to release the diol product. Asp132 acts as a base to deprotonate a water molecule. The hydroxide ion then performs a nucleophilic attack at one of the ring carbons, forcing epoxide ring opening. At the same time, Asp101 donates a proton to the oxygen atom of the epoxide ring to encourage the ring opening. Each carboxylate group of Asp132 and Asp101 maintains a pair of hydrogen bonds with guanidino group of Arg99 during the reaction and Arg99 acts as a proton shuttle to reverse the charge of Asp132 and Asp101 after the reaction. Tyr53 and Asn55 also help activate the water molecule for nucleophilic attack by hydrogen bond donation and acceptance respectively and also have roles in stabilising the transition state and positioning of the reactants.

Catalytic Residues Roles

UniProt PDB* (1nww)
Tyr53 Tyr53A As part of an Asn-Tyr diad with Asn 55, acts to position water correctly for nucleophilic attack whilst increasing its nucleophilicity through hydrogen bonding and is also important in stabilising the transition state. electrostatic stabiliser
Asn55 (main-C) Asn55A (main-C) As part of Asn-Tyr diad with Tyr 53, activates water through hydrogen bonding from its main chain carbonyl to allow nucleophilic attack on the epoxide and also important in stabilising the transition state. electrostatic stabiliser
Arg99 Arg99A As the central residue in the Asp Arg Asp triad is able to alter the pKas of the two Asp residues (Asp 101 and Asp 132) so that they can act as proton donor and acceptor respectively. This allows the unique push pull mechanism of the hydrolase to take place. proton relay, proton acceptor, electrostatic stabiliser, proton donor
Asp101 Asp101A Protonates the oxygen atom of the epoxide, thus facilitating the opening of the ring and simultaneous nucleophilic attack from water. proton acceptor, proton donor
Asp132 Asp132A Activates water by deprotonation to allow it to act as a nucleophile and attack the epoxide. activator, proton acceptor, proton donor
*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

overall reactant used, overall product formed, bimolecular nucleophilic substitution, proton transfer, inferred reaction step, native state of enzyme regenerated, proton relay

References

  1. Lind ME et al. (2013), Angew Chem Int Ed Engl, 52, 4563-4567. Quantum chemistry as a tool in asymmetric biocatalysis: limonene epoxide hydrolase test case. DOI:10.1002/anie.201300594. PMID:23512539.
  2. Rinaldi S et al. (2018), ACS Catal, 8, 5698-5707. Understanding Complex Mechanisms of Enzyme Reactivity: The Case of Limonene-1,2-Epoxide Hydrolases. DOI:10.1021/acscatal.8b00863.
  3. Hou QQ et al. (2012), Biochim Biophys Acta, 1824, 263-268. QM/MM study of the mechanism of enzymatic limonene 1,2-epoxide hydrolysis. DOI:10.1016/j.bbapap.2011.08.014. PMID:21925621.
  4. Arand M et al. (2003), EMBO J, 22, 2583-2592. Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site. DOI:10.1093/emboj/cdg275. PMID:12773375.

Step 1. Asp132 activates the water molecule by abstracting a proton. This results in the nucleophilic attack of the epoxide on the substrate. Simultaneously, Asp101 also protonates the epoxide group of the substrate. Other residues shown are involved in the positioning of the reactants, water and limonene-1,2- diol. This results in the formation of limonene-1,2- diol.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr53A electrostatic stabiliser
Arg99A electrostatic stabiliser
Asn55A (main-C) electrostatic stabiliser
Asp132A activator
Asp101A proton donor
Asp132A proton acceptor

Chemical Components

overall reactant used, overall product formed, ingold: bimolecular nucleophilic substitution, proton transfer

Step 2. Proton transfer via Arg99 to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg99A proton relay, proton donor
Asp101A proton acceptor
Asp132A proton donor
Arg99A proton acceptor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated, proton relay
Download: Image, Marvin File

Step 1. Asp132 activates the water molecule by abstracting a proton. This results in the nucleophilic attack of the epoxide on the substrate. Simultaneously, Asp101 also protonates the epoxide group of the substrate. Other residues shown are involved in the positioning of the reactants, water and limonene-1,2- diol. This results in the formation of limonene-1,2- diol.

Step 2. Proton transfer via Arg99 to regenerate the active site.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Morwenna Hall, Noa Marson