NADPH dehydrogenase

 

NADPH dehydrogenase, or old yellow enzyme (OYE), isolated from Candida albicans, is a NADPH oxidoreductase. Its FMN cofactor (in plants the cofactor is FAD) is reduced by NADPH, and in turn goes on to reduce the substrate. The exact physiological substrate(s) of OYE are yet to be confirmed, though it is known to act on molecular oxygen, alpha,beta-unsaturated ketones and aldehydes, and quinones. OYE is thought to be involved in general detoxification within the cell.

 

Reference Protein and Structure

Sequence
Q02899 UniProt (1.6.99.1) IPR001155 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces pastorianus (Brewer's Yeast) Uniprot
PDB
1oya - OLD YELLOW ENZYME AT 2 ANGSTROMS RESOLUTION: OVERALL STRUCTURE, LIGAND BINDING AND COMPARISON WITH RELATED FLAVOPROTEINS (2.0 Å) PDBe PDBsum 1oya
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1oya)
Cofactors
Fmnh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.6.99.1)

hydron
CHEBI:15378ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
+
cyclohex-2-enone
CHEBI:15977ChEBI
→
cyclohexanone
CHEBI:17854ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
Alternative enzyme names: NADPH diaphorase, NADPH-dehydrogenase, NADPH-diaphorase, NADPH(2) diaphorase, NADPH(2)-dehydrogenase, OYE, TPNH dehydrogenase, TPNH-diaphorase, Diaphorase, Dihydronicotinamide adenine dinucleotide phosphate dehydrogenase, Old yellow enzyme, Reduced nicotinamide adenine dinucleotide phosphate dehydrogenase, Triphosphopyridine diaphorase, Triphosphopyridine nucleotide diaphorase, NADPH:(acceptor) oxidoreductase,

Enzyme Mechanism

Introduction

This mechanism describes the reduction of an alpha,beta-unsaturated ketone. NADPH transfers a hydride from C4 to the si face of the N5 position of FMN, reducing it and causing a negative charge to build up on N1. Reduced FMN then transfers a hydride from the N5 position to the beta-carbon of the substrate, forming an enolate intermediate that is stabilised by His191, Arg194 and Tyr196. The carbonyl reforms and the alpha-carbon is protonated by Tyr196 to form the saturated carbonyl product.

Catalytic Residues Roles

UniProt PDB* (1oya)
Asn252 Asn251(252)A Activates Tyr196 to act as a general acid/base. hydrogen bond donor, increase acidity
His192 His191(192)A His191 polarises the carbonyl bond of the substrate, thus making alpha,beta-unsaturated ketone more electrophilic. It also stabilises the enolate intermediate by hydrogen bonding to the negatively charged oxygen. hydrogen bond donor, electrostatic stabiliser
Asn195 Asn194(195)A Asn194 polarises the carbonyl bond of the substrate, thus making alpha,beta-unsaturated ketone more electrophilic. It also stabilises the enolate intermediate by hydrogen bonding to the negatively charged oxygen. hydrogen bond donor, electrostatic stabiliser
Thr38 Thr37(38)A Thr37 decreases the redox potential of the FMN cofactor by hydrogen bonding to the C4 carbonyl. hydrogen bond donor, electrostatic stabiliser
Tyr197 Tyr196(197)A Tyr196 donates a proton to C-alpha of the enolate intermediate to form the saturated ketone product. It is also thought to be able to stabilise the transition state state for the transfer of hydride by interacting with C-alpha. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, 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, intermediate formation, overall reactant used, overall product formed, cofactor used, rate-determining step, proton transfer, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Kohli RM et al. (1998), J Biol Chem, 273, 32763-32770. The Oxidative Half-reaction of Old Yellow Enzyme. THE ROLE OF TYROSINE 196. DOI:10.1074/jbc.273.49.32763. PMID:9830020.
  2. Brown BJ et al. (2002), J Biol Chem, 277, 2138-2145. The Role of Glutamine 114 in Old Yellow Enzyme. DOI:10.1074/jbc.m108453200. PMID:11668181.
  3. Xu D et al. (1999), Proc Natl Acad Sci U S A, 96, 3556-3561. The role of threonine 37 in flavin reactivity of the old yellow enzyme. DOI:10.1073/pnas.96.7.3556. PMID:10097075.
  4. Brown BJ et al. (1998), J Biol Chem, 273, 32753-32762. On the Active Site of Old Yellow Enzyme. ROLE OF HISTIDINE 191 AND ASPARAGINE 194. DOI:10.1074/jbc.273.49.32753. PMID:9830019.
  5. Fox KM et al. (1994), Structure, 2, 1089-1105. Old yellow enzyme at 2 A resolution: overall structure, ligand binding, and comparison with related flavoproteins. DOI:10.2210/pdb1oyb/pdb. PMID:7881908.

Step 1. NADPH reduces the FMN cofactor. NADPH transfers a hydride from the C4 atom to the si face of the N5 position of FMN [PMID:9830020].

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Catalytic Residues Roles

Residue Roles
Thr37(38)A hydrogen bond donor, electrostatic stabiliser
Tyr196(197)A electrostatic stabiliser, hydrogen bond donor
Asn251(252)A hydrogen bond donor

Chemical Components

hydride transfer, intermediate formation, overall reactant used, overall product formed, cofactor used, rate-determining step

Step 2. The anionic FMN intermediate acts as a hydride donor to the a-b unsaturated substrate. Tyr196 acts as the proton donor in the trans reduction of the substrate double bond. The exact physiological substrate(s) of NADPH dehydrogenase are yet to be confirmed, though it is known to act on molecular oxygen, alpha,beta-unsaturated ketones, aldehydes, and quinones. Reduction across a double bond occurs in a stereospecific trans mechanism [PMID:11668181, PMID:9830020].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn194(195)A hydrogen bond donor, electrostatic stabiliser
His191(192)A hydrogen bond donor, electrostatic stabiliser
Thr37(38)A hydrogen bond donor
Tyr196(197)A hydrogen bond acceptor, hydrogen bond donor
Asn251(252)A increase acidity, hydrogen bond donor
Tyr196(197)A proton donor

Chemical Components

hydride transfer, proton transfer, native state of cofactor regenerated, overall product formed, overall reactant used

Step 3. The Tyr196A is reprotonated by a solvent molecule.

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Catalytic Residues Roles

Residue Roles
Tyr196(197)A hydrogen bond donor, hydrogen bond acceptor
Thr37(38)A hydrogen bond donor
Asn251(252)A hydrogen bond donor
Tyr196(197)A proton acceptor

Chemical Components

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

Step 1. NADPH reduces the FMN cofactor. NADPH transfers a hydride from the C4 atom to the si face of the N5 position of FMN [PMID:9830020].

Step 2. The anionic FMN intermediate acts as a hydride donor to the a-b unsaturated substrate. Tyr196 acts as the proton donor in the trans reduction of the substrate double bond. The exact physiological substrate(s) of NADPH dehydrogenase are yet to be confirmed, though it is known to act on molecular oxygen, alpha,beta-unsaturated ketones, aldehydes, and quinones. Reduction across a double bond occurs in a stereospecific trans mechanism [PMID:11668181, PMID:9830020].

Step 3. The Tyr196A is reprotonated by a solvent molecule.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Alex Gutteridge, Craig Porter, Amelia Brasnett