5-carboxymethyl-2-hydroxymuconate Delta-isomerase

 

5-carboxymethyl-2-hydroxymuconate isomerase (CHMI) transforms 5-carboxymethyl-2-hydroxy-muconic acid into 5-oxo-pent-3-ene-1,2,5-tricarboxylic acid during the third step of the homoprotocatechuate catabolic pathway [PMID:2194841]. CHMI is a member of the tautomerase superfamily many of which utilise a unique N-terminal proline residue as a general acid/base.

 

Reference Protein and Structure

Sequence
Q05354 UniProt (5.3.3.10) IPR004220 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
1otg - 5-CARBOXYMETHYL-2-HYDROXYMUCONATE ISOMERASE (2.1 Å) PDBe PDBsum 1otg
Catalytic CATH Domains
3.30.429.10 CATHdb (see all for 1otg)
Click To Show Structure

Enzyme Reaction (EC:5.3.3.10)

(2R,3E)-5-oxopent-3-ene-1,2,5-tricarboxylate
CHEBI:87491ChEBI
→
(2E,4Z)-5-hydroxypenta-2,4-diene-1,2,5-tricarboxylate(3-)
CHEBI:47961ChEBI
Alternative enzyme names: CHM isomerase, 5-carboxymethyl-2-hydroxymuconic acid isomerase,

Enzyme Mechanism

Introduction

Pro2 acts as a base abstracting a proton from C3 causing an tautomerisation to occur with a ene-diolate intermediate being formed. The intermediate collapses with a rearrangement of the double bonds and Pro2 protonates O6. The hydrophobic residue Pro36 modifies the pKa of Pro2 allowing it to acts as an acid-base catalyst. Two arginine residues stabilize the carboxylate side chains of the substrate.

Catalytic Residues Roles

UniProt PDB* (1otg)
Pro2 Pro2(1)A Acts as a general acid/base. proton acceptor, proton donor
Arg72, Arg41 Arg72(71)A, Arg41(40)A Helps stabilise the reactive intermediates and transition states, as well as being part of the substrate binding site. electrostatic stabiliser
Pro36 Pro36(35)A Due to similarity with other tautomerase superfamily members (most notable 4-OT), this residue is thought to perturbate the pKa of the N-terminal proline so that it is able to act as a general acid/base. modifies pKa
*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, assisted keto-enol tautomerisation, proton transfer, electron transfer, overall product formed, native state of enzyme regenerated

References

  1. Subramanya HS et al. (1996), Biochemistry, 35, 792-802. Enzymatic Ketonization of 2-Hydroxymuconate:  Specificity and Mechanism Investigated by the Crystal Structures of Two Isomerases†. DOI:10.1021/bi951732k. PMID:8547259.
  2. Johnson WH et al. (1995), J Am Chem Soc, 117, 8719-8726. Stereochemical Studies of 5-(Carboxymethyl)-2-hydroxymuconate Isomerase and 5-(Carboxymethyl)-2-oxo-3-hexene-1,6-dioate Decarboxylase from Escherichia coli C: Mechanistic and Evolutionary Implications. DOI:10.1021/ja00139a004.
  3. Roper DI et al. (1990), FEBS Lett, 266, 63-66. Purification, some properties and nucleotide sequence of 5-carboxymethyl-2-hydroxymuconate isomerase of Escherichia coli C. PMID:2194841.

Step 1. Pro2 acts as a base which causes a keto-enol tautomerisation.

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Catalytic Residues Roles

Residue Roles
Pro36(35)A modifies pKa
Arg41(40)A electrostatic stabiliser
Arg72(71)A electrostatic stabiliser
Pro2(1)A proton acceptor

Chemical Components

overall reactant used, assisted keto-enol tautomerisation, proton transfer

Step 2. The ene-diolate intermediate collapses and there is a rearrangement of the double bonds which allows O6 to accept a proton from the proline.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg72(71)A electrostatic stabiliser
Arg41(40)A electrostatic stabiliser
Pro36(35)A modifies pKa
Pro2(1)A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, electron transfer, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Pro2 acts as a base which causes a keto-enol tautomerisation.

Step 2. The ene-diolate intermediate collapses and there is a rearrangement of the double bonds which allows O6 to accept a proton from the proline.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday, James Willey