Dihydroorotate oxidase (class II)

 

Dihydroororate reductase in humans catalyses a key step in the synthesis of pyrimidines, as it is able to convert dihydroororate into ororate, using FMN and Ubiquinone as cofactors for the reaction. The human enzyme is part of family 2, with homology to other mammalian dihydroororate reductases and to the equivalent enzymes in bacteria which are clustered in family 1. The enzyme is particularly important in T cells because of their high nucleotide turnover through new DNA synthesis.

 

Reference Protein and Structure

Sequence
Q02127 UniProt (1.3.5.2) IPR005719 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1d3g - HUMAN DIHYDROOROTATE DEHYDROGENASE COMPLEXED WITH BREQUINAR ANALOG (1.6 Å) PDBe PDBsum 1d3g
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1d3g)
Cofactors
Fmnh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.3.5.2)

ubiquinones
CHEBI:16389ChEBI
+
(S)-dihydroorotate
CHEBI:30864ChEBI
→
ubiquinol
CHEBI:17976ChEBI
+
orotate
CHEBI:30839ChEBI
Alternative enzyme names: Dihydroorotate:ubiquinone oxidoreductase, (S)-dihydroorotate:(acceptor) oxidoreductase, (S)-dihydroorotate:acceptor oxidoreductase, DHODH, DHOD, DHOdehase,

Enzyme Mechanism

Introduction

In this step-wise proposal, the reaction proceeds via an enolate intermediate, which is stabilised by two highly conserved asparagines. The reaction proceeds via initial abstraction of a proton from dihydroororate by Ser 215 which is activated to act as an acid base by Thr 218 and Phe 149. This creates an oxyanion which collapses in a second step to transfers a hydride ion to FMN to form FMNH-. The hydride ion is then transferred to Ubiquinone which associates with the inner mitochondrial membrane and thus contributes to the electron transfer chain.

Catalytic Residues Roles

UniProt PDB* (1d3g)
Asn283, Asn216, Asn144 Asn284(255)A, Asn217(188)A, Asn145(116)A Help bind and stabilise the intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Thr217 Thr218(189)A Through electrostatic contacts, is able to lower the pKa of Ser 215 to allow it to act as a general base. It has also been suggested that this residue might act as a proton relay between the bulk solvent and the active site. activator, hydrogen bond acceptor, enhance reactivity, electrostatic stabiliser
Phe148 Phe149(120)A Through contacts between the pi electron ring and the OH group of Ser 215, is able to lower the pKa of Ser 215 sufficiently for it to act as a general base. activator, electrostatic stabiliser, polar/non-polar interaction
Lys254 Lys255(226)A Stabilises the FMN after the transfer of a hydride ion, possibly acts as a general acid/base to allow the reduction to be completed. hydrogen bond donor, electrostatic stabiliser
Ser214 Ser215(186)A Acts to remove proton from the dihydroororate substrate, thus allowing the carbanion to form which can transfer a hydride to the FMN cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, polar/non-polar interaction, proton relay
*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, hydride transfer, assisted keto-enol tautomerisation, aromatic unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, overall reactant used, native state of cofactor regenerated, intermediate terminated, overall product formed, inferred reaction step, native state of enzyme regenerated, cofactor used, native state of cofactor is not regenerated

References

  1. Fagan RL et al. (2006), Biochemistry, 45, 14926-14932. Mechanism of Flavin Reduction in Class 2 Dihydroorotate Dehydrogenases†. DOI:10.1021/bi060919g. PMID:17154530.
  2. Alves CN et al. (2015), Phys Chem Chem Phys, 17, 17790-17796. Insights into the mechanism of oxidation of dihydroorotate to orotate catalysed by human class 2 dihydroorotate dehydrogenase: a QM/MM free energy study. DOI:10.1039/c5cp02016f. PMID:26087682.
  3. Kow RL et al. (2009), Biochemistry, 48, 9801-9809. Disruption of the Proton Relay Network in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli. DOI:10.1021/bi901024m. PMID:19694481.
  4. Fagan RL et al. (2009), Biochemistry, 48, 7169-7178. Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli. DOI:10.1021/bi900370s. PMID:19530672.
  5. Baumgartner R et al. (2006), J Med Chem, 49, 1239-1247. Dual Binding Mode of a Novel Series of DHODH Inhibitors. DOI:10.1021/jm0506975. PMID:16480261.

Step 1. Ser215 (activated through a triad with water and Thr218) abstracts a proton from the substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr218(189)A enhance reactivity
Asn145(116)A electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser
Ser215(186)A proton donor, proton acceptor, proton relay

Chemical Components

proton transfer

Step 2. The enolate intermediate collapses, eliminating a hydride ion which adds onto the FMN.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn145(116)A electrostatic stabiliser
Phe149(120)A electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Thr218(189)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser
Lys255(226)A electrostatic stabiliser

Chemical Components

hydride transfer, assisted keto-enol tautomerisation

Step 3. The product dissociates from the active site, to be replaced by ubiquinone. The reduced FMN eliminates the hydride ion which is added to the ubiquinone, initiating double bond rearrangement and deprotonation of Ser215 via a water molecue to generate ubiquinol in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn284(255)A electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Phe149(120)A polar/non-polar interaction, activator
Ser215(186)A hydrogen bond donor, hydrogen bond acceptor, polar/non-polar interaction
Thr218(189)A hydrogen bond acceptor, activator
Lys255(226)A hydrogen bond donor, electrostatic stabiliser
Ser215(186)A proton acceptor, proton relay, proton donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, hydride transfer, proton transfer, overall reactant used, native state of cofactor regenerated, intermediate terminated, overall product formed, inferred reaction step, native state of enzyme regenerated, cofactor used, native state of cofactor is not regenerated
Download: Image, Marvin File

Step 1. Ser215 (activated through a triad with water and Thr218) abstracts a proton from the substrate.

Step 2. The enolate intermediate collapses, eliminating a hydride ion which adds onto the FMN.

Step 3. The product dissociates from the active site, to be replaced by ubiquinone. The reduced FMN eliminates the hydride ion which is added to the ubiquinone, initiating double bond rearrangement and deprotonation of Ser215 via a water molecue to generate ubiquinol in an inferred return step.

Products.

Introduction

In this step-wise proposal, a hydride transfer from the substrate occurs first and the reaction proceeds via an iminium intermediate. In a second step, abstraction of a proton from dihydroororate by Ser 215 which is activated to act as an acid base by Thr 218 and Phe 149 occurs to produce the final product. The hydride ion is then transferred to Ubiquinone which associates with the inner mitochondrial membrane and thus contributes to the electron transfer chain.

Catalytic Residues Roles

UniProt PDB* (1d3g)
Asn283, Asn216, Asn144 Asn284(255)A, Asn217(188)A, Asn145(116)A Help bind and stabilise the intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Thr217 Thr218(189)A Through electrostatic contacts, is able to lower the pKa of Ser 215 to allow it to act as a general base. It has also been suggested that this residue might act as a proton relay between the bulk solvent and the active site. activator, hydrogen bond acceptor, enhance reactivity, electrostatic stabiliser
Phe148 Phe149(120)A Through contacts between the pi electron ring and the OH group of Ser 215, is able to lower the pKa of Ser 215 sufficiently for it to act as a general base. activator, enhance reactivity, electrostatic stabiliser, polar/non-polar interaction
Lys254 Lys255(226)A Stabilises the FMN after the transfer of a hydride ion, possibly acts as a general acid/base to allow the reduction to be completed. hydrogen bond donor, electrostatic stabiliser
Ser214 Ser215(186)A Acts to remove proton from the dihydroororate substrate, thus allowing the carbanion to form which can transfer a hydride to the FMN cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, polar/non-polar interaction, proton donor, proton relay, 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

hydride transfer, proton transfer, assisted tautomerisation (not keto-enol), aromatic unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, overall reactant used, native state of cofactor regenerated, intermediate terminated, overall product formed, inferred reaction step, native state of enzyme regenerated, cofactor used, native state of cofactor is not regenerated

References

  1. Fagan RL et al. (2006), Biochemistry, 45, 14926-14932. Mechanism of Flavin Reduction in Class 2 Dihydroorotate Dehydrogenases†. DOI:10.1021/bi060919g. PMID:17154530.
  2. Alves CN et al. (2015), Phys Chem Chem Phys, 17, 17790-17796. Insights into the mechanism of oxidation of dihydroorotate to orotate catalysed by human class 2 dihydroorotate dehydrogenase: a QM/MM free energy study. DOI:10.1039/c5cp02016f. PMID:26087682.
  3. Kow RL et al. (2009), Biochemistry, 48, 9801-9809. Disruption of the Proton Relay Network in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli. DOI:10.1021/bi901024m. PMID:19694481.
  4. Fagan RL et al. (2009), Biochemistry, 48, 7169-7178. Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli. DOI:10.1021/bi900370s. PMID:19530672.

Step 1. The substrate eliminates a hydride ion, which adds to FMN.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys255(226)A electrostatic stabiliser
Phe149(120)A enhance reactivity, polar/non-polar interaction
Asn145(116)A electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser

Chemical Components

hydride transfer

Step 2. Ser215 abstracts a proton from the intermediate to form the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe149(120)A electrostatic stabiliser
Thr218(189)A electrostatic stabiliser
Lys255(226)A electrostatic stabiliser
Thr218(189)A enhance reactivity
Asn145(116)A electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser
Ser215(186)A proton acceptor, proton donor, proton relay

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol)

Step 3. The product dissociates from the active site, to be replaced by ubiquinone. The reduced FMN eliminates the hydride ion which is added to the ubiquinone, initiating double bond rearrangement and deprotonation of Ser215 via a water molecue to generate ubiquinol in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe149(120)A polar/non-polar interaction, activator
Ser215(186)A hydrogen bond donor, hydrogen bond acceptor, polar/non-polar interaction
Thr218(189)A hydrogen bond acceptor, activator
Lys255(226)A hydrogen bond donor, electrostatic stabiliser
Ser215(186)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser
Ser215(186)A proton acceptor, proton relay, proton donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, hydride transfer, proton transfer, overall reactant used, native state of cofactor regenerated, intermediate terminated, overall product formed, inferred reaction step, native state of enzyme regenerated, cofactor used, native state of cofactor is not regenerated
Download: Image, Marvin File

Step 1. The substrate eliminates a hydride ion, which adds to FMN.

Step 2. Ser215 abstracts a proton from the intermediate to form the final product.

Step 3. The product dissociates from the active site, to be replaced by ubiquinone. The reduced FMN eliminates the hydride ion which is added to the ubiquinone, initiating double bond rearrangement and deprotonation of Ser215 via a water molecue to generate ubiquinol in an inferred return step.

Products.

Introduction

This mechanism proposal represents the concerted reaction. The reaction proceeds via initial abstraction of a proton from dihydroororate by Ser 215 which is activated to act as an acid base by Thr 218 and Phe 149. This creates a carbanion which transfers a hydride ion to FMN to form FMNH2, assisted by protonation of the FMN by Lys 255, forming the product. The hydride ion is then transferred to Ubiquinone which associates with the inner mitochondrial membrane and thus contributes to the electron transfer chain.

Catalytic Residues Roles

UniProt PDB* (1d3g)
Asn283, Asn216, Asn144 Asn284(255)A, Asn217(188)A, Asn145(116)A Help bind and stabilise the intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Thr217 Thr218(189)A Through electrostatic contacts, is able to lower the pKa of Ser 215 to allow it to act as a general base. It has also been suggested that this residue might act as a proton relay between the bulk solvent and the active site. activator, hydrogen bond acceptor
Phe148 Phe149(120)A Through contacts between the pi electron ring and the OH group of Ser 215, is able to lower the pKa of Ser 215 sufficiently for it to act as a general base. activator, electrostatic stabiliser, polar/non-polar interaction
Lys254 Lys255(226)A Stabilises the FMN after the transfer of a hydride ion, possibly acts as a general acid/base to allow the reduction to be completed. hydrogen bond donor, electrostatic stabiliser
Ser214 Ser215(186)A Acts to remove proton from the dihydroororate substrate, thus allowing the carbanion to form which can transfer a hydride to the FMN cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, polar/non-polar interaction, proton relay
*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

bimolecular elimination, aromatic bimolecular nucleophilic addition, hydride transfer, overall reactant used, cofactor used, intermediate formation, overall product formed, aromatic unimolecular elimination by the conjugate base, proton transfer, native state of cofactor regenerated, intermediate terminated, inferred reaction step, native state of enzyme regenerated, native state of cofactor is not regenerated

References

  1. Liu S et al. (2000), Structure, 8, 25-33. Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents. DOI:10.1016/s0969-2126(00)00077-0. PMID:10673429.
  2. Kow RL et al. (2009), Biochemistry, 48, 9801-9809. Disruption of the Proton Relay Network in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli. DOI:10.1021/bi901024m. PMID:19694481.
  3. Fagan RL et al. (2009), Biochemistry, 48, 7169-7178. Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli. DOI:10.1021/bi900370s. PMID:19530672.
  4. Baumgartner R et al. (2006), J Med Chem, 49, 1239-1247. Dual Binding Mode of a Novel Series of DHODH Inhibitors. DOI:10.1021/jm0506975. PMID:16480261.

Step 1. Ser215 deprotonates the C5 of dihydroorotate, eliminating a hydride ion, which is added to FMN.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe149(120)A polar/non-polar interaction, electrostatic stabiliser
Ser215(186)A hydrogen bond acceptor, polar/non-polar interaction
Thr218(189)A hydrogen bond acceptor
Lys255(226)A hydrogen bond donor
Asn145(116)A electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser
Ser215(186)A proton acceptor, proton donor, proton relay

Chemical Components

ingold: bimolecular elimination, ingold: aromatic bimolecular nucleophilic addition, hydride transfer, overall reactant used, cofactor used, intermediate formation, overall product formed

Step 2. The product dissociates from the active site, to be replaced by ubiquinone. The reduced FMN eliminates the hydride ion which is added to the ubiquinone, initiating double bond rearrangement and deprotonation of Ser215 via a water molecue to generate ubiquinol in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe149(120)A polar/non-polar interaction, activator
Ser215(186)A hydrogen bond donor, hydrogen bond acceptor, polar/non-polar interaction
Thr218(189)A hydrogen bond acceptor, activator
Lys255(226)A hydrogen bond donor, electrostatic stabiliser
Asn217(188)A electrostatic stabiliser
Asn284(255)A electrostatic stabiliser
Ser215(186)A proton acceptor, proton relay, proton donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, hydride transfer, proton transfer, overall reactant used, native state of cofactor regenerated, intermediate terminated, overall product formed, inferred reaction step, native state of enzyme regenerated, cofactor used, native state of cofactor is not regenerated
Download: Image, Marvin File

Step 1. Ser215 deprotonates the C5 of dihydroorotate, eliminating a hydride ion, which is added to FMN.

Step 2. The product dissociates from the active site, to be replaced by ubiquinone. The reduced FMN eliminates the hydride ion which is added to the ubiquinone, initiating double bond rearrangement and deprotonation of Ser215 via a water molecue to generate ubiquinol in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Sophie T. Williams, Peter Sarkies