2,4-dienoyl-CoA reductase (NADPH)

 

2,4-dienoyl-CoA reductase (DCR) in E. coli is an iron-sulfur flavoenzyme which contains FMN, FAD, and a 4Fe-4S cluster. It is also a monomer, unlike that of its eukaryotic counterparts which form homotetramers and lack the flavin and iron-sulfur cofactors. DCR utilises NADPH to remove the C4-C5 double bond of unsaturated fatty acids. DCR is unusual in that it lacks stereospecificity, catalysing the reduction of both natural fatty acids with cis double bonds, as well as substrates containing trans double bonds, this might be explained by the large size of the substrate binding pocket [PMID:12840019].

 

Reference Protein and Structure

Sequence
P42593 UniProt (1.3.1.34) IPR001155 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ps9 - The Crystal Structure and Reaction Mechanism of E. coli 2,4-Dienoyl CoA Reductase (2.2 Å) PDBe PDBsum 1ps9
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1ps9)
Cofactors
Fadh2(2-) (1), Fmnh2(2-) (1), Tetra-mu3-sulfido-tetrairon (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.3.1.34)

4,5-saturated-trans-2-enoyl-CoA(4-)
CHEBI:85100ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
→
trans,trans-2,4-dienoyl-CoA(4-)
CHEBI:85101ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
Alternative enzyme names: 4-enoyl coenzyme A (reduced nicotinamide adenine dinucleotide phosphate) reductase, 4-enoyl-CoA reductase, 4-enoyl-CoA reductase (NADPH(2)), 4-enoyl-CoA reductase (NADPH), 2,4-dienoyl-CoA reductase (NADPH),

Enzyme Mechanism

Introduction

The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. The fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr and His are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and completes the reaction.

Catalytic Residues Roles

UniProt PDB* (1ps9)
His253 His252A The imidazole group of His-252, which is within hydrogen bonding distance to Tyr-166 (2.7 A), is likely to stabilise the phenolate ion formed during catalysis. hydrogen bond donor, electrostatic stabiliser
Arg215 Arg214A The reduced FMN may exist in anionic state during reduction as has been proposed, suggesting that Arg-214 would be ideally positioned to provide a counter ion. hydrogen bond donor, electrostatic stabiliser
Gln340 Gln339A Tthe amide group of the Gln-339 side chain may participate in electron transfer between FAD and the iron-sulfur cluster by dividing electron transfer between the two cofactors from one large through-space transfer to two smaller through-space steps. However, the FAD to 4Fe-4S distance is sufficiently short that this is unlikely to be essential. single electron relay, hydrogen bond acceptor, hydrogen bond donor, single electron acceptor, single electron donor
Glu165 Glu164A Glu-164 forms a hydrogen bond to the thioester carbonyl oxygen atom of the acyl chain, which helps to promote the positive character of the C5 atom of the acyl chain through resonance of the conjugated double bonds. hydrogen bond donor, electrostatic stabiliser
Tyr167 Tyr166A Tyr-166 acts as the catalytic residue that protonates the C4 atom during substrate reduction. hydrogen bond acceptor, hydrogen bond donor, 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

aromatic unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, hydride transfer, overall reactant used, intermediate formation, overall product formed, electron transfer, proton transfer, radical formation, cofactor used, native state of cofactor regenerated, electron relay, radical termination, bimolecular nucleophilic addition, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Hubbard PA et al. (2003), J Biol Chem, 278, 37553-37560. The Crystal Structure and Reaction Mechanism of Escherichia coli 2,4-Dienoyl-CoA Reductase. DOI:10.1074/jbc.m304642200. PMID:12840019.

Step 1. NADP eliminates a hydride ion, which is added to FAD. The nicotinamide ring of NADP interacts with the re-face of the isoalloxazine ring, resulting in fully reduced FAD [PMID:12840019].

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Catalytic Residues Roles

Residue Roles
Glu164A hydrogen bond donor
Tyr166A hydrogen bond acceptor, hydrogen bond donor
Arg214A hydrogen bond donor
His252A hydrogen bond donor
Gln339A hydrogen bond acceptor, hydrogen bond donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, hydride transfer, overall reactant used, intermediate formation, overall product formed

Step 2. FAD transfers a single electron, through Gln339 and an iron-sulfur cluster, to FMN, which deprotonates water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu164A hydrogen bond donor
Tyr166A hydrogen bond acceptor, hydrogen bond donor
Arg214A hydrogen bond donor
His252A hydrogen bond donor
Gln339A hydrogen bond acceptor, hydrogen bond donor
Gln339A single electron relay, single electron donor, single electron acceptor

Chemical Components

electron transfer, proton transfer, radical formation, overall reactant used, intermediate formation, cofactor used, native state of cofactor regenerated, electron relay

Step 3. Water deprotonates FAD, which initiates a second single electron transfer, through Gln339 and an iron-sulfur cluster, to FMN. This regenerates the FAD cofactor, and produces fully reduced FMN.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu164A hydrogen bond donor
Tyr166A hydrogen bond acceptor, hydrogen bond donor
Arg214A hydrogen bond donor
His252A hydrogen bond donor
Gln339A hydrogen bond acceptor, hydrogen bond donor
Gln339A single electron relay, single electron donor, single electron acceptor

Chemical Components

proton transfer, electron transfer, radical termination, intermediate formation, native state of cofactor regenerated, electron relay

Step 4. FMN eliminates a hydride ion, which is added to the C5 of the substrate. This initiates double bond rearrangement, resulting in an oxyanion on the C1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu164A hydrogen bond donor
Tyr166A hydrogen bond acceptor, hydrogen bond donor
Arg214A hydrogen bond donor, electrostatic stabiliser
His252A hydrogen bond donor
Gln339A hydrogen bond acceptor, hydrogen bond donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: bimolecular nucleophilic addition, hydride transfer, overall reactant used, native state of cofactor regenerated, intermediate formation

Step 5. The oxyanion collapses, initiating double bond rearrangement, the C4 of the intermediate deprotonates Tyr166.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu164A hydrogen bond donor, electrostatic stabiliser
Tyr166A hydrogen bond acceptor, hydrogen bond donor
Arg214A hydrogen bond donor
His252A hydrogen bond donor
Gln339A hydrogen bond acceptor, hydrogen bond donor
Tyr166A proton donor

Chemical Components

proton transfer, intermediate terminated, overall product formed

Step 6. Tyr166 deprotonates water in an inferred return step.

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Catalytic Residues Roles

Residue Roles
Tyr166A hydrogen bond acceptor
Arg214A hydrogen bond donor
His252A hydrogen bond donor, electrostatic stabiliser
Gln339A hydrogen bond acceptor, hydrogen bond donor
Tyr166A proton acceptor

Chemical Components

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

Step 1. NADP eliminates a hydride ion, which is added to FAD. The nicotinamide ring of NADP interacts with the re-face of the isoalloxazine ring, resulting in fully reduced FAD [PMID:12840019].

Step 2. FAD transfers a single electron, through Gln339 and an iron-sulfur cluster, to FMN, which deprotonates water.

Step 3. Water deprotonates FAD, which initiates a second single electron transfer, through Gln339 and an iron-sulfur cluster, to FMN. This regenerates the FAD cofactor, and produces fully reduced FMN.

Step 4. FMN eliminates a hydride ion, which is added to the C5 of the substrate. This initiates double bond rearrangement, resulting in an oxyanion on the C1.

Step 5. The oxyanion collapses, initiating double bond rearrangement, the C4 of the intermediate deprotonates Tyr166.

Step 6. Tyr166 deprotonates water in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Alex Gutteridge, Craig Porter