Kynureninase

 

Kynureninase is a pyridoxal-5'-phosphate (PLP)-dependent enzyme sourced from Pseudomonas fluorescens that catalyses the hydrolytic cleavage of L-kynurenine to anthranilic acid and L-alanine. This reaction is a key step in the catabolism of L-tryptophan. In fungi and vertebrates, kynurenine is first hydroxylated to 3-hydroxykynurenine - the preferred substrate of kynureninase in those organisms, resulting in 3-hydroxyanthranilate and L-alanine. Kynureninase also plays a role in the biosynthesis of NAD-(P)+. The product of kynureninase is subsequently converted by 3-hydroxyanthranilate-3,4-dioxygenase to quinolinic acid - a precursor of NAD-(P)+. Quinolinic acid is a neurotoxin due to it's agonist effects on N-methyl-D-aspartate receptor. Excessive levels of quinolinic acid have been implicated in a wide range of neurological disorders such as epilepsy, stroke and AIDS related dementia. Therefore, inhibitors of kynureninase are of interest as potential drugs for the treatment of these CNS disorders.

 

Reference Protein and Structure

Sequence
P83788 UniProt (3.7.1.3) IPR010111 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas fluorescens (Bacteria) Uniprot
PDB
1qz9 - The Three Dimensional Structure of Kynureninase from Pseudomonas fluorescens (1.85 Å) PDBe PDBsum 1qz9
Catalytic CATH Domains
3.40.640.10 CATHdb 3.90.1150.10 CATHdb (see all for 1qz9)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:3.7.1.3)

water
CHEBI:15377ChEBI
+
L-kynurenine zwitterion
CHEBI:57959ChEBI
→
hydron
CHEBI:15378ChEBI
+
L-alanine zwitterion
CHEBI:57972ChEBI
+
anthranilate
CHEBI:16567ChEBI

Enzyme Mechanism

Introduction

Lys 227 binds PLP to Kynureninase. Electrophilicity of PLP is enhanced through close contact with Asp 201 which aids protonation of the pyridine ring. Phe 129 is Pi-stacked on top of the pyridine ring of PLP, stabilising the cofactor.The phosphate of PLPis protonated by Tyr226 which then accepts a proton from L-kynureninethus forming a hydrogen bond netewrok between PLP, Tyr226 and L-kynurenine. L-kynurenine displaces the Lys 227, binding to PLP, forming kynurenine external aldimine. Lys 227 deprotonates the alpha-carbon of the kynurenine external aldimine. Lys 227 protonates the C-4' of the quinonoid intermediate. Lys 227 deprotonates a water molecule, which performs nucleophilic attack upon the gamma-carbonyl of the ketimine intermediate. This forms the gem-diolate intermediate via deprotonation of Tyr226. The gem-diolate intermediate collapses, cleaving bond between the beta-carbon and gamma-carbon. Lys 227 protonates the beta-carbon of the enamine intermediate. Lys 227 deprotonates C-4' of the pyruvate ketimine intermediate. Lys 227 protonates the alanine quinonoid intermediate, giving alanine external aldimine. Lys 227 displaces L-alanine, releasing it.

Catalytic Residues Roles

UniProt PDB* (1qz9)
Tyr226 Tyr226A Mediates proton transfer between the substrate and the phosphate, which accelerates formation of external aldimine and gem-diol intermediates hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Arg375 Arg375A Stabilises the negative charge of the carboxyl group on the substrate electrostatic stabiliser
Phe129 Phe129A Phe 129 is Pi-stacked on top of the pyridine ring of PLP, stabilising the cofactor. electrostatic stabiliser
Asp201 Asp201A Asp 201 aids protonation of the pyridine ring, enhancing the electrophilicity of PLP. electrostatic stabiliser
Lys227 Lys227A 1. Lys 227 binds PLP to Kynureninase. 2. Lys 227 deprotonates the alpha-carbon of the kynurenine external aldimine. 3. Lys 227 protonates the C-4' of the quinonoid intermediate. 4. Lys 227 deprotonates a water molecule, which performs nucleophilic attack upon the gamma-carbonyl of the ketimine intermediate. 5. Lys 227 protonates the beta-carbon of the enamine intermediate. 6. Lys 227 deprotonates C-4' of the pyruvate ketimine intermediate. 7. Lys 227 protonates the alanine quinonoid intermediate, giving alanine external aldimine. 8. Lys 227 displaces L-alanine, releasing it. nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor
*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, bimolecular nucleophilic substitution, overall reactant used, intermediate formation, cofactor used, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, bimolecular nucleophilic addition, overall product formed, rate-determining step, inferred reaction step, native state of enzyme regenerated

References

  1. Momany C et al. (2004), Biochemistry, 43, 1193-1203. Three-Dimensional Structure of Kynureninase fromPseudomonas fluorescens†,‡. DOI:10.1021/bi035744e. PMID:14756555.
  2. Phillips RS et al. (2014), FEBS J, 281, 1100-1109. The phosphate of pyridoxal-5'-phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase. DOI:10.1111/febs.12671. PMID:24304904.
  3. Phillips RS (2014), Arch Biochem Biophys, 544, 69-74. Structure and mechanism of kynureninase. DOI:10.1016/j.abb.2013.10.020. PMID:24200862.

Step 1. The phosphate of PLP accepts a proton from Tyr226 which then accepts a proton from the amine group of L-kynurenine which then nucleophilically attacks the carbon of the C-N bond between PLP and Lys227 and as a result releases Lys227.

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Catalytic Residues Roles

Residue Roles
Asp201A electrostatic stabiliser
Phe129A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A proton donor, proton acceptor, proton relay
Lys227A electron pair acceptor

Chemical Components

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

Step 2. The secondary amine that results from the initial attack initiates an elimination of the covalently bound lysine, resulting in free PLP and lysine in a neutral state.

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A hydrogen bond acceptor, hydrogen bond donor
Lys227A nucleofuge

Chemical Components

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

Step 3. Lys227 deprotonates the CH adjacent to the bound amine, resulting in double bond rearrangement as the PLP acts as an electron sink.

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A hydrogen bond acceptor, hydrogen bond donor
Lys227A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The PLP feeds the electrons back, resulting in the C=C attached to the aromatic ring deprotonating Lys227

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A hydrogen bond acceptor, hydrogen bond donor
Lys227A proton donor

Chemical Components

proton transfer, intermediate formation

Step 5. Lys227 then abstracts a proton from water so that it can nucleophilically attack the carbon of the carbonyl in the Kyurenine ketimine intermediate. The oxygen of the carbonyl accepts a proton from Tyr226 which produces the gemdiol intermediate. Tyr266 then accepts a proton from the phosphate group.

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Lys227A proton acceptor
Tyr226A proton relay, proton donor, proton acceptor

Chemical Components

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

Step 6. Tyr226 is deprotonated by the phosphate group of PLP so that in turn it can then deprotonate the hydroxyl group of the gemdiol intermediate which results in the cleavage of anthranilate from the enamine intermediate.

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Catalytic Residues Roles

Residue Roles
Arg375A electrostatic stabiliser
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Tyr226A hydrogen bond acceptor, hydrogen bond donor
Tyr226A proton acceptor, proton donor, proton relay

Chemical Components

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

Step 7. The enamine accepts a proton on its beta carbon.

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A hydrogen bond donor, hydrogen bond acceptor
Lys227A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation

Step 8. Lys227 deprotonates the CH adjacent to the amine, resulting in double bond rearrangement as PLP acts as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A hydrogen bond acceptor, hydrogen bond donor
Lys227A proton acceptor

Chemical Components

proton transfer, intermediate formation, rate-determining step

Step 9. The PLP feeds the electrons back, resulting in the N=C bond deprotonating Lys227.

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Tyr226A hydrogen bond acceptor, hydrogen bond donor
Lys227A proton donor

Chemical Components

proton transfer, intermediate formation

Step 10. Lys227 nucleophilically attacks the carbon of the C=N bond which results in the cleavage of L-alanine from PLP and its substitution for Lys227 and the proton on the phosphate group is lost to the environment which returns the active site back to its native state.

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Catalytic Residues Roles

Residue Roles
Phe129A electrostatic stabiliser
Asp201A electrostatic stabiliser
Arg375A electrostatic stabiliser
Lys227A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, inferred reaction step, intermediate collapse, native state of enzyme regenerated, overall product formed
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Step 1. The phosphate of PLP accepts a proton from Tyr226 which then accepts a proton from the amine group of L-kynurenine which then nucleophilically attacks the carbon of the C-N bond between PLP and Lys227 and as a result releases Lys227.

Step 2. The secondary amine that results from the initial attack initiates an elimination of the covalently bound lysine, resulting in free PLP and lysine in a neutral state.

Step 3. Lys227 deprotonates the CH adjacent to the bound amine, resulting in double bond rearrangement as the PLP acts as an electron sink.

Step 4. The PLP feeds the electrons back, resulting in the C=C attached to the aromatic ring deprotonating Lys227

Step 5. Lys227 then abstracts a proton from water so that it can nucleophilically attack the carbon of the carbonyl in the Kyurenine ketimine intermediate. The oxygen of the carbonyl accepts a proton from Tyr226 which produces the gemdiol intermediate. Tyr266 then accepts a proton from the phosphate group.

Step 6. Tyr226 is deprotonated by the phosphate group of PLP so that in turn it can then deprotonate the hydroxyl group of the gemdiol intermediate which results in the cleavage of anthranilate from the enamine intermediate.

Step 7. The enamine accepts a proton on its beta carbon.

Step 8. Lys227 deprotonates the CH adjacent to the amine, resulting in double bond rearrangement as PLP acts as an electron sink.

Step 9. The PLP feeds the electrons back, resulting in the N=C bond deprotonating Lys227.

Step 10. Lys227 nucleophilically attacks the carbon of the C=N bond which results in the cleavage of L-alanine from PLP and its substitution for Lys227 and the proton on the phosphate group is lost to the environment which returns the active site back to its native state.

Products.

Contributors

Fiona J. E. Morgan, Gemma L. Holliday, Charity Hornby