Cytokinin dehydrogenase

 

Cytokinin dehydrogenase (CKX) from Zea mays catalyses the irreversible oxidation of cytokinins, a type of plant hormone which promotes cell division and differentiation. CKX catalyses the conversion of N6-(delta2-isopentenyl)-adenine into adenine. Increased enzyme activity causes drastic abnormalities in the growth and development of the plant.

 

Reference Protein and Structure

Sequence
Q9T0N8 UniProt (1.5.99.12) IPR015345 (Sequence Homologues) (PDB Homologues)
Biological species
Zea mays (Maize) Uniprot
PDB
1w1o - Native Cytokinin Dehydrogenase (1.7 Å) PDBe PDBsum 1w1o
Catalytic CATH Domains
3.30.465.10 CATHdb 3.30.43.10 CATHdb 3.40.462.10 CATHdb (see all for 1w1o)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.5.99.12)

N(6)-dimethylallyladenine
CHEBI:17660ChEBI
+
water
CHEBI:15377ChEBI
+
1,4-benzoquinone
CHEBI:16509ChEBI
→
adenine
CHEBI:16708ChEBI
+
3-methylbut-2-enal
CHEBI:15825ChEBI
+
hydroquinone
CHEBI:17594ChEBI
Alternative enzyme names: Cytokinin oxidase, N(6)-dimethylallyladenine:(acceptor) oxidoreductase, 6-N-dimethylallyladenine:acceptor oxidoreductase,

Enzyme Mechanism

Introduction

The mechanism proceeds as follows:

  1. A hydride is accepted by FAD from the substrate C11 atom.
  2. A standard FADH oxidation then occurs with quinone.
  3. The imine is then hydrolysed in a non-enzymatic step.

Catalytic Residues Roles

UniProt PDB* (1w1o)
His105 His105A Is covalently bound to cofactor FAD. covalently attached, alter redox potential
Asp169 Asp169A Stabilises the positive charge on the intermediate. hydrogen bond acceptor, electrostatic stabiliser
Glu288 Glu288A Forms a carboxylate-carboxylate pair with Asp 169, facilitating the stabilization of the intermediate by Asp 169. activator, hydrogen bond 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

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

References

  1. Malito E et al. (2004), J Mol Biol, 341, 1237-1249. Structures of Michaelis and Product Complexes of Plant Cytokinin Dehydrogenase: Implications for Flavoenzyme Catalysis. DOI:10.1016/j.jmb.2004.06.083. PMID:15321719.
  2. Galuszka P et al. (2005), Plant Cell Physiol, 46, 716-728. Tissue Localization of Cytokinin Dehydrogenase in Maize: Possible Involvement of Quinone Species Generated from Plant Phenolics by Other Enzymatic Systems in the Catalytic Reaction. DOI:10.1093/pcp/pci074. PMID:15746157.
  3. Frébortová J et al. (2004), Biochem J, 380, 121-130. Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors. DOI:10.1042/bj20031813. PMID:14965342.

Step 1. The nitrogen of trimethylamine initiates the elimination of a hydride ion, which is added to FAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His105A covalently attached
Asp169A hydrogen bond acceptor
Glu288A hydrogen bond donor
His105A alter redox potential

Chemical Components

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

Step 2. The first single electron transfer from FAD to quinone. The semi-quinone undergoes double bond rearrangement that results in the deprotonation of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His105A covalently attached
Asp169A hydrogen bond acceptor, electrostatic stabiliser
Glu288A hydrogen bond donor, activator
His105A alter redox potential

Chemical Components

electron transfer, proton transfer, radical formation, overall reactant used, intermediate formation

Step 3. The second single electron transfer from FAD to quinone which is facilitated by the abstraction of a proton from the FMN by water. The quinol deprotonates another water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His105A covalently attached
Asp169A hydrogen bond acceptor, electrostatic stabiliser
Glu288A hydrogen bond donor, activator
His105A alter redox potential

Chemical Components

electron transfer, proton transfer, radical termination, native state of cofactor regenerated, intermediate terminated, overall product formed, native state of enzyme regenerated

Step 4. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce adenine and 3-methylbut-2-enal.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, proton transfer, ingold: bimolecular nucleophilic addition

Step 5. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce adenine and 3-methylbut-2-enal.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: intramolecular elimination, reaction occurs outside the enzyme, proton transfer
Download: Image, Marvin File

Step 1. The nitrogen of trimethylamine initiates the elimination of a hydride ion, which is added to FAD.

Step 2. The first single electron transfer from FAD to quinone. The semi-quinone undergoes double bond rearrangement that results in the deprotonation of water.

Step 3. The second single electron transfer from FAD to quinone which is facilitated by the abstraction of a proton from the FMN by water. The quinol deprotonates another water molecule.

Step 4. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce adenine and 3-methylbut-2-enal.

Step 5. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce adenine and 3-methylbut-2-enal.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Ellie Wright