Glycine C-acetyltransferase

 

Threonine is degraded into glycine and the acetyl group in a pathway common to prokaryotic and eukaryotic cells. The pathway is two steps long; the first step is the oxidation of the hydroxy group of threonine to create 2-amino-3-ketobutyrate by L-threonine dehydrogenase. The second step is catalysed by 2-amino-3-ketobutyrate CoA ligase (KBL); this is a PLP-dependent acetyltransferase, transferring the newly formed acetyl group of the substrate to Coenzyme A to give glycine and acetyl-CoA. The two enzymes needed for the pathway form a complex; this is because the aminoketobutyrate intermediate (substrate for KBL) spontaneously decarboxylates in aqueous solution.

 

Reference Protein and Structure

Sequence
P0AB77 UniProt (2.3.1.29) IPR011282 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1fc4 - 2-AMINO-3-KETOBUTYRATE COA LIGASE (2.0 Å) PDBe PDBsum 1fc4
Catalytic CATH Domains
3.40.640.10 CATHdb (see all for 1fc4)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:2.3.1.29)

coenzyme A(4-)
CHEBI:57287ChEBI
+
L-2-amino-3-oxobutanoic acid
CHEBI:40673ChEBI
→
glycine
CHEBI:15428ChEBI
+
acetyl-CoA(4-)
CHEBI:57288ChEBI
Alternative enzyme names: 2-amino-3-ketobutyrate CoA ligase, 2-amino-3-ketobutyrate coenzyme A ligase, 2-amino-3-ketobutyrate-CoA ligase, Aminoacetone synthase, Glycine acetyltransferase, Aminoacetone synthetase, KBL, AKB ligase,

Enzyme Mechanism

Introduction

Lys 244 has a Schiff base linkage with the PLP cofactor. It is displaced by the amino acid substrate, in the usual fashion to give an external aldimine (PLP and threonine with a Schiff base linkage). In these steps His 213 acts as a general acid to the phenolic oxygen of PLP (with the phenolic proton being transferred to Lys 244), and a general base to the amino group of the joining threonine. The thiol of CoA is nucleophilic and attacks the carbon of the carbonyl group of the aldimine. This gives a tetrahedral intermediate where the oxyanion is stabilised by hydrogen bonding to Ser 185. Lys 244 deprotonates the sulphur atom. The tetrahedral intermediate collapses to release acetyl-CoA. The glycine moiety and PLP are held in a quinoid intermediate. Lys 244 protonates the nitrogen of the glycine moiety, regenerating the substrate-PLP Schiff base link. Lys 244 displaces the group bound to PLP in the usual fashion (His 213 again acting as a general acid/base) to release glycine and regenerate the original active PLP-enzyme adduct.

Catalytic Residues Roles

UniProt PDB* (1fc4)
Ser185 Ser185(188)A Forms a weak hydrogen bond with O3 of the oxyanion tetrahedral intermediate. hydrogen bond donor, electrostatic stabiliser
His213 His213(216)A His 213 acts as a general acid and base during the steps where the PLP Schiff base linkage is transferred from Lys 244 to the amino acid, and vice versa. proton acceptor, proton donor
Lys244 Lys244(247)A Lys 244 is Schiff base-linked to the PLP cofactor in the resting state, and displaces the glycine product in the last step of the reaction.
Lys 244 deprotonates the thiol after the formation of the tetrahedral intermediate. The proton is transferred to the nitrogen atom of the glycine moiety of the quinoid intermediate to regenerate the imine linkage. 		covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, proton donor, proton acceptor, electrostatic stabiliser, electron pair acceptor, electron pair 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

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

References

  1. Schmidt A et al. (2001), Biochemistry, 40, 5151-5160. Three-Dimensional Structure of 2-Amino-3-ketobutyrate CoA Ligase fromEscherichia coliComplexed with a PLP−Substrate Intermediate:  Inferred Reaction Mechanism. DOI:10.1021/bi002204y. PMID:11318637.
  2. Mann S et al. (2011), Biochim Biophys Acta, 1814, 1459-1466. Pyridoxal-5′-phosphate-dependent enzymes involved in biotin biosynthesis: Structure, reaction mechanism and inhibition. DOI:10.1016/j.bbapap.2010.12.004. PMID:21182990.

Step 1. His213 acts as an acid catalyst for the transaldimation reaction. The amine of the substrate L-2-amino-3-oxobutanoic acid attacks the PLP cofactor in a nucleophilic addition and the bound Lys244 deprotonates the newly attached amine.

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Catalytic Residues Roles

Residue Roles
Lys244(247)A covalently attached, hydrogen bond donor
His213(216)A proton donor
Lys244(247)A proton acceptor, electron pair acceptor

Chemical Components

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

Step 2. His213 accepts a proton from the secondary amine that results from the initial attack which also 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
Lys244(247)A nucleofuge
His213(216)A proton acceptor

Chemical Components

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

Step 3. CoA thiol group attacks PLP-substrate to form a tetrahedral intermediate in which Lys244 removes the excess proton. Ser185 stabilises oxyanion intermediate.

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Catalytic Residues Roles

Residue Roles
Ser185(188)A hydrogen bond donor, electrostatic stabiliser
Lys244(247)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate collapse

Step 4. Unimolecular elimination to form acetyl-CoA , which is released and also a quinoid intermediate, PLP acts an electron sink.

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Catalytic Residues Roles

Residue Roles
Ser185(188)A electrostatic stabiliser, hydrogen bond donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, heterolysis, overall product formed

Step 5. Lys244 protonates the quinoid intermediate, PLP feeds an electron pair back to form the glycine external aldimine.

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Catalytic Residues Roles

Residue Roles
Lys244(247)A hydrogen bond donor, electrostatic stabiliser, proton donor

Chemical Components

intermediate collapse, proton transfer

Step 6. In the reverse reaction, this is the first step of the His213 catalysed Lys244 displacing the product glycine.

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Catalytic Residues Roles

Residue Roles
Lys244(247)A covalently attached
His213(216)A proton donor
Lys244(247)A nucleophile

Chemical Components

proton transfer, intermediate collapse

Step 7. Unimolecular elimination to release the product glycine.

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Catalytic Residues Roles

Residue Roles
Lys244(247)A covalently attached, electron pair donor, proton donor

Chemical Components

overall product formed, ingold: unimolecular elimination by the conjugate base, intermediate terminated, intermediate collapse, schiff base formed

Step 8. Proton transfer from PLP to His213 to regenerate active site.

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Catalytic Residues Roles

Residue Roles
Lys244(247)A hydrogen bond acceptor, covalently attached
His213(216)A proton acceptor
Lys244(247)A proton donor

Chemical Components

proton transfer, proton relay, intermediate terminated, native state of enzyme regenerated
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Step 1. His213 acts as an acid catalyst for the transaldimation reaction. The amine of the substrate L-2-amino-3-oxobutanoic acid attacks the PLP cofactor in a nucleophilic addition and the bound Lys244 deprotonates the newly attached amine.

Step 2. His213 accepts a proton from the secondary amine that results from the initial attack which also initiates an elimination of the covalently bound lysine, resulting in free PLP and lysine in a neutral state.

Step 3. CoA thiol group attacks PLP-substrate to form a tetrahedral intermediate in which Lys244 removes the excess proton. Ser185 stabilises oxyanion intermediate.

Step 4. Unimolecular elimination to form acetyl-CoA , which is released and also a quinoid intermediate, PLP acts an electron sink.

Step 5. Lys244 protonates the quinoid intermediate, PLP feeds an electron pair back to form the glycine external aldimine.

Step 6. In the reverse reaction, this is the first step of the His213 catalysed Lys244 displacing the product glycine.

Step 7. Unimolecular elimination to release the product glycine.

Step 8. Proton transfer from PLP to His213 to regenerate active site.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Morwenna Hall