Deoxyhypusine synthase

 

Deoxyhypusine is a highly rare posttranslational modification that occurs in eukaryotes where the it is vital for the formation of the initiation factor eIF5A. The enzyme carrying the reaction out is deoxyhypusine synthase which is able to transfer a butylamine moiety from spermidine to a lysine in eIF5A, using NAD as a cofactor. Since active eIF5A is required for tumour cell proliferation, design of inhibitors for this enzyme could prove useful in the treatment of certain cancers. The high specificity of the enzyme is also of interest, because the only incidence of this modification in eukaryotic proteins appears to be in this particular case.

 

Reference Protein and Structure

Sequence
P49366 UniProt (2.5.1.46) IPR002773 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1roz - Deoxyhypusine synthase holoenzyme in its low ionic strength, high pH crystal form (2.21 Å) PDBe PDBsum 1roz
Catalytic CATH Domains
3.40.910.10 CATHdb (see all for 1roz)
Cofactors
Nadh(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.46)

L-lysinium residue
CHEBI:29969ChEBI
+
spermidine(3+)
CHEBI:57834ChEBI
→
trimethylenediaminium
CHEBI:57484ChEBI
+
deoxyhypusine(2+) residue
CHEBI:82657ChEBI
Alternative enzyme names: [eIF-5A]-deoxyhypusine synthase, Spermidine dehydrogenase, (4-aminobutyl)lysine synthase, Spermidine:eIF5A-lysine 4-aminobutyltransferase (propane-1,3-diamine-forming),

Enzyme Mechanism

Introduction

The enzymatic reaction proceeds in four steps. First, hydride transfer from spermidine to NAD with concomitant deprotonation by His 288 leads to the dehydration of spermidine to dehydrospermidine. The dehydrospermidine is thus activated to act as an electrophile; nucleophilic attack from lys 239 transfers the butylimine moiety to the enzyme forming an imine-enzyme intermediate, releasing diaminopropane. The lysine residue from eIF5A to be modified then attacks as a nucleophile, resulting in the formation of eIF5A imine intermediate and regenerating the catalytic lysine residue. Finally, the imine is converted to the butylamine moiety to form deoxyhypusine and NAD. Hydride transfer to and from NAD is assisted by Glu 157 which activates C4 of the NAD through electrostatic interactions.

Catalytic Residues Roles

UniProt PDB* (1roz)
Lys329 Lys329A Acts as a nucleophile to accept the butylimine moiety from dehydrospermidine, forming an enzyme-imine intermediate. This then acts as an electrophile for transfer of the butylimine moiety to eIF5A with the lysine residue in eIF5A to be modified acting as a nucleophile. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor, electron pair donor
His288 His288A Accepts proton from spermidine to facilitate hydride transfer. Subsequently releases proton back to the modified lysine in eIF5A to allow hydride transfer from NADH to occur. proton acceptor, proton donor
Glu137 Glu137B(BA) Activates the NAD towards accepting a hydride ion in the first stage of the reaction through repulsion between the rozC4's pi electrons and the negative charge. Subsequently activates the eIF5A towards accepting a hydride ion through repulsion between the CN double bond and the negative charge. 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

bimolecular nucleophilic addition, cofactor used, hydride transfer, intermediate formation, overall reactant used, proton transfer, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated, overall product formed

References

  1. Umland TC et al. (2004), J Biol Chem, 279, 28697-28705. A New Crystal Structure of Deoxyhypusine Synthase Reveals the Configuration of the Active Enzyme and of an EnzymeNADInhibitor Ternary Complex. DOI:10.1074/jbc.m404095200. PMID:15100216.
  2. Park MH (2006), J Biochem, 139, 161-169. The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A). DOI:10.1093/jb/mvj034. PMID:16452303.
  3. Wolff EC et al. (2000), J Biol Chem, 275, 9170-9177. Deoxyhypusine Synthase Generates and Uses Bound NADH in a Transient Hydride Transfer Mechanism. DOI:10.1074/jbc.275.13.9170. PMID:10734052.

Step 1. Spermidine transfers a hydride to NAD with concomitant deprotonation by His 288 leads to the dehydration of spermidine to dehydrospermidine.

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Catalytic Residues Roles

Residue Roles
Glu137B(BA) electrostatic stabiliser
His288A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, cofactor used, hydride transfer, intermediate formation, overall reactant used, proton transfer

Step 2. Lys329 nucleophilically attacks the carbon the C=N bond which will then accept a proton from Lys329 on the nitrogen of the double bond.

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Catalytic Residues Roles

Residue Roles
Glu137B(BA) electrostatic stabiliser
Lys329A covalently attached
Lys329A nucleophile, proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, cofactor used, enzyme-substrate complex formation, intermediate formation

Step 3. The secondary amine that results from the initial attack initiates an elimination of diaminopropane and produces an imine-enzyme intermediate. Deprotonation of Lys329 by water is inferred.

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Catalytic Residues Roles

Residue Roles
Glu137B(BA) electrostatic stabiliser
Lys329A covalently attached, proton donor, electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, intermediate formation

Step 4. The lysine residue from eIF5A to be modified then attacks as a nucleophile on the carbon of the C=N bond. The double bond will then accept a proton from the eIF5A lysine.

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Catalytic Residues Roles

Residue Roles
Glu137B(BA) electrostatic stabiliser
Lys329A covalently attached
Lys329A proton acceptor, electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, overall reactant used

Step 5. The secondary amine produced initiates an elimination which releases Lys329 which when released will accept a proton from the substrate nitrogen.

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Catalytic Residues Roles

Residue Roles
Glu137B(BA) electrostatic stabiliser
Lys329A proton acceptor, nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, proton transfer

Step 6. NADH eliminates a hydride form its C4 position to the carbon of the C=N bond of the intermediate resulting in the concomitant protonation of the nitrogen of the C=N bond by His288 producing the final product.

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Catalytic Residues Roles

Residue Roles
Glu137B(BA) electrostatic stabiliser
His288A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, hydride transfer, ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Spermidine transfers a hydride to NAD with concomitant deprotonation by His 288 leads to the dehydration of spermidine to dehydrospermidine.

Step 2. Lys329 nucleophilically attacks the carbon the C=N bond which will then accept a proton from Lys329 on the nitrogen of the double bond.

Step 3. The secondary amine that results from the initial attack initiates an elimination of diaminopropane and produces an imine-enzyme intermediate. Deprotonation of Lys329 by water is inferred.

Step 4. The lysine residue from eIF5A to be modified then attacks as a nucleophile on the carbon of the C=N bond. The double bond will then accept a proton from the eIF5A lysine.

Step 5. The secondary amine produced initiates an elimination which releases Lys329 which when released will accept a proton from the substrate nitrogen.

Step 6. NADH eliminates a hydride form its C4 position to the carbon of the C=N bond of the intermediate resulting in the concomitant protonation of the nitrogen of the C=N bond by His288 producing the final product.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Charity Hornby