Beta-lysine 5,6-aminomutase

 

Lysine 5,6-aminomutase (5,6-LAM) is sourced from Clostridium sticklandii. It catalyses the reversible transformations of D-lysine into 2,5-diaminohexanoate and of L-beta-lysine into 3,5-diaminohexanoate. The activity of 5,6-LAM is dependent on pyridoxal-5'-phosphate (PLP) and adenosylcobalamin.

 

Reference Protein and Structure

Sequences
E3PRJ5 UniProt (5.4.3.3)
E3PRJ4 UniProt (5.4.3.3) IPR006158 (Sequence Homologues) (PDB Homologues)
Biological species
Acetoanaerobium sticklandii DSM 519 (Bacteria) Uniprot
PDB
1xrs - Crystal structure of Lysine 5,6-Aminomutase in complex with PLP, cobalamin, and 5'-deoxyadenosine (2.8 Å) PDBe PDBsum 1xrs
Catalytic CATH Domains
3.40.50.280 CATHdb 3.20.20.440 CATHdb (see all for 1xrs)
Cofactors
Cob(iii)alamin (1), Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:5.4.3.3)

(3S)-3,6-diammoniohexanoate
CHEBI:57434ChEBI
→
(3S,5S)-3,5-diammoniohexanoate
CHEBI:57436ChEBI
Alternative enzyme names: Beta-lysine mutase, L-beta-lysine 5,6-aminomutase, Beta-lysine 5,6-aminomutase, D-lysine 5,6-aminomutase, D-alpha-lysine mutase, Adenosylcobalamin-dependent D-lysine 5,6-aminomutase,

Enzyme Mechanism

Introduction

The substrate Lys displaces Lys 144 from PLP with a standard transaldimination reaction. Adenosylcobalamin (AdoCbl) homolytically cleaves to form Ado-CH2 radical. Ado-CH2 radical accepts a proton and an electron from the substrate Lys delta carbon. A three membered ring is formed between the delta and epsilon carbons and the nitrogen of the substrate Lys, breaking the double bond, leaving just a single bond between the substrate N and PLP C atoms, and a radical PLP. The double bond between the nitrogen of the substrate Lys and the carbon of PLP is reformed, homolytically cleaving the N-C-epsilon bond of the substrate Lys. Ado-CH3 then donates a proton and an electron back to the product, reforming the Ado-CH2 radical. Lys 144 then nucleophilically attacks PLP, to displace the product with a standard transaldimination reaction.

Catalytic Residues Roles

UniProt PDB* (1xrs)
Lys144 Lys144B Lys 144 is covalently attached to PLP. As Lys substrate binds to PLP, Lys 144 accepts a pair of electrons from PLP, breaking the Lys 144 - PLP bond. When the product is formed, Lys 144 acts as a nucleophile and attacks PLP forcing the product to leave. electron pair acceptor, electron pair donor, nucleofuge, nucleophile
Tyr266 Tyr263A Stabilizes radical intermediates. radical stabiliser
Lys373, Asp301 Lys370A, Asp298A Form salt bridges to the substrate lysine to facilitate the initial transaldimination and formation of the 5'-deoxyadenosyl radical. promote homolysis
*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, intermediate formation, schiff base formed, homolysis, overall reactant used, intermediate collapse, radical formation, intramolecular elimination, radical propagation, tautomerisation (not keto-enol), intramolecular homolytic addition, intramolecular homolytic substitution, radical termination, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated, intermediate terminated

References

  1. Tang KH et al. (2003), Arch Biochem Biophys, 418, 49-54. Kinetic and biochemical analysis of the mechanism of action of lysine 5,6-aminomutase. DOI:10.1016/s0003-9861(03)00346-1. PMID:13679082.
  2. Lo HH et al. (2016), Chem Commun (Camb), 52, 6399-6402. The molecular mechanism of the open-closed protein conformational cycle transitions and coupled substrate binding, activation and product release events in lysine 5,6-aminomutase. DOI:10.1039/c6cc01888b. PMID:27086547.
  3. Chen YH et al. (2013), J Am Chem Soc, 135, 788-794. Mechanism-based inhibition reveals transitions between two conformational states in the action of lysine 5,6-aminomutase: a combination of electron paramagnetic resonance spectroscopy, electron nuclear double resonance spectroscopy, and density functional theory study. DOI:10.1021/ja309603a. PMID:23231091.
  4. Chen YH et al. (2011), J Am Chem Soc, 133, 17152-17155. Radical stabilization is crucial in the mechanism of action of lysine 5,6-aminomutase: role of tyrosine-263α as revealed by electron paramagnetic resonance spectroscopy. DOI:10.1021/ja207766c. PMID:21939264.

Step 1. The reaction starts with nucleophilic attack from the substrate to the internal aldimine bond between PLP and Lys 144. Forming an intermediate.

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Catalytic Residues Roles

Residue Roles
Lys144B electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 2. A new aldimine bond is formed between the substrate lysine and PLP, this leads to the cleavage of Lys 144 from PLP. Simultaneously to lysine transaldimination, homolytic cleavage of the Co-C bond occurs creating a 5' -deoxyadenosyl radical and cob(II)alamin. Lys 370 and and Asp 298 form a salt bridge with the alpha carboxylate and amino group, respectively, of the substrate . These interactions are required for these two reactions to occur.

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Catalytic Residues Roles

Residue Roles
Asp298A promote homolysis
Lys370A promote homolysis
Lys144B nucleofuge

Chemical Components

schiff base formed, homolysis, overall reactant used, intermediate collapse, radical formation, ingold: intramolecular elimination

Step 3. Hydrogen is abstracted by 5' -dAdo from the substrate generating deoxyadenosine and the substrate related radical.

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Catalytic Residues Roles

Residue Roles

Chemical Components

radical propagation

Step 4. A reversible tautomerization occurs generating a more stable radical which is stabilized by Tyr 263.

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Catalytic Residues Roles

Residue Roles
Tyr263A radical stabiliser

Chemical Components

tautomerisation (not keto-enol)

Step 5. Intramolecular radical isomerization occurs generating a cyclic aziridincarbinyl-PLP radical. Which is again stabilized by Tyr263.

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Catalytic Residues Roles

Residue Roles
Tyr263A radical stabiliser

Chemical Components

ingold: intramolecular homolytic addition

Step 6. Further radical isomerization occurs leading to collapse of the cyclic aziridincarbinyl-PLP radical.

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Catalytic Residues Roles

Residue Roles
Tyr263A radical stabiliser

Chemical Components

ingold: intramolecular homolytic substitution

Step 7. The substrate abstracts a hydrogen radical from 5'-deoxyadenosine, forming a 5'-deoxyadenosyl radical. This leaves what will become the product bound to PLP.

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Catalytic Residues Roles

Residue Roles

Chemical Components

radical propagation

Step 8. There is nucleophilic attack from Lys 144 to the aldimine bond between the product and PLP. The transaldimination of lysines occurs simultaneously with the formation of the Co-C leading to radical termination, similarly to the first lysine substitution.

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Catalytic Residues Roles

Residue Roles
Lys144B nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, radical termination, native state of cofactor regenerated

Step 9. The aldimine bond between Lys144 and PLP is reformed. This leads to the product being cleaved from PLP.

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Catalytic Residues Roles

Residue Roles
Lys144B electron pair donor

Chemical Components

ingold: intramolecular elimination, overall product formed, native state of enzyme regenerated, intermediate terminated, schiff base formed
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Step 1. The reaction starts with nucleophilic attack from the substrate to the internal aldimine bond between PLP and Lys 144. Forming an intermediate.

Step 2. A new aldimine bond is formed between the substrate lysine and PLP, this leads to the cleavage of Lys 144 from PLP. Simultaneously to lysine transaldimination, homolytic cleavage of the Co-C bond occurs creating a 5' -deoxyadenosyl radical and cob(II)alamin. Lys 370 and and Asp 298 form a salt bridge with the alpha carboxylate and amino group, respectively, of the substrate . These interactions are required for these two reactions to occur.

Step 3. Hydrogen is abstracted by 5' -dAdo from the substrate generating deoxyadenosine and the substrate related radical.

Step 4. A reversible tautomerization occurs generating a more stable radical which is stabilized by Tyr 263.

Step 5. Intramolecular radical isomerization occurs generating a cyclic aziridincarbinyl-PLP radical. Which is again stabilized by Tyr263.

Step 6. Further radical isomerization occurs leading to collapse of the cyclic aziridincarbinyl-PLP radical.

Step 7. The substrate abstracts a hydrogen radical from 5'-deoxyadenosine, forming a 5'-deoxyadenosyl radical. This leaves what will become the product bound to PLP.

Step 8. There is nucleophilic attack from Lys 144 to the aldimine bond between the product and PLP. The transaldimination of lysines occurs simultaneously with the formation of the Co-C leading to radical termination, similarly to the first lysine substitution.

Step 9. The aldimine bond between Lys144 and PLP is reformed. This leads to the product being cleaved from PLP.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, James Willey