2,2-dialkylglycine decarboxylase (pyruvate)

 

Dialkylglycine decarboxylase (DGD) is a pyridoxal 5-phosphate (PLP) dependent enzyme which catalyses the decarboxylation dependent transamination between dialkylglycine and pyruvate. The dialkylglycine is decarboxylated to give CO2 and a dialkylketone whilst the amino group is transferred to the PLP cofactor from where it reacts with pyruvate to produce L-alanine.

The resting state of most PLP dependent enzyme has the PLP linked covalently via a Schiff base to a lysine residue, referred to as the internal aldimime. The amino group of the substrate then reacts to replace the lysine, forming the external aldimime adduct. The PLP pyridoxal ring acts as an electron sink in the reaction.

DGD has been shown by structural analysis to be a member of the type I fold family. The central PLP domain of the subunits (most members are dimers or oligomers with dihedral symmetry, DGD is a tetramer) is an alpha/beta structure with a unique seven stranded beta sheet.

 

Reference Protein and Structure

Sequence
P16932 UniProt (4.1.1.64) IPR005814 (Sequence Homologues) (PDB Homologues)
Biological species
Burkholderia cepacia (Pseudomonas cepacia) Uniprot
PDB
1d7r - CRYSTAL STRUCTURE OF THE COMPLEX OF 2,2-DIALKYLGLYCINE DECARBOXYLASE WITH 5PA (2.0 Å) PDBe PDBsum 1d7r
Catalytic CATH Domains
3.40.640.10 CATHdb 3.90.1150.10 CATHdb (see all for 1d7r)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:4.1.1.64)

2,2-dialkylglycine zwitterion
CHEBI:57689ChEBI
+
hydron
CHEBI:15378ChEBI
+
pyruvate
CHEBI:15361ChEBI
→
L-alanine zwitterion
CHEBI:57972ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
+
dialkyl ketone
CHEBI:18044ChEBI
Alternative enzyme names: 2,2-dialkyl-2-amino acid-pyruvate aminotransferase, Alpha-dialkyl amino acid transaminase, L-alanine-alpha-ketobutyrate aminotransferase, Dialkyl amino acid (pyruvate) decarboxylase, Dialkylamino-acid decarboxylase (pyruvate), 2,2-dialkylglycine carboxy-lyase (amino-transferring),

Enzyme Mechanism

Introduction

DGD is an unusual PLP dependent enzyme in that it catalyses both decarboxylation and transamination reactions in its ping pong catalytic cycle. The first half reaction is an oxidative decarboxylation. The external aldimime is formed by a transamination reaction where the substrate alpha amino group displaces the E-amino group of Lys272. This intermediate is activated towards decarboxylation through interactions with Arg406 and Glu210 which hold the external aldimine is an orientation which maximises overlap between the pi orbitals of the conjugated system, stabilising the transition state and therefore accelerating the rate of bond cleavage. Loss of carbon dioxide gives the quinoid, which on protonation of C(4) forms a ketimine intermediate. This is hydrolysed, resulting in the pyridoxamine form of DGD (DGD-PMP) and a ketone. The catalytic cycle is then completed by a classical transamination half-reaction between DGD-PMP and an alpha keto-acid, preferably pyruvate regenerating the aldehyde form of the coenzyme and giving an L-amino acid product.

Catalytic Residues Roles

UniProt PDB* (1d7r)
Trp138 Trp138A Ensures that the un-bound cofactor is held in the correct orientation in the active site. steric role
Asp243 Asp243A Acts to stabilise the PLP intermediate, activating it so that it can act as an electron sink. increase electrophilicity, electrostatic stabiliser
Gln246 Gln246A Helps stabilise the reactive intermediates and transition states. electrostatic stabiliser
Lys272 Lys272A Lys272 is covalently bound to the PLP cofactor in the ground state of the enzyme, it also acts as a general acid/base and catalytic nucleophile. covalent catalysis, proton shuttle (general acid/base)
Glu210, Arg406 Glu210A, Arg406A Arg406 and Glu210 hold the external aldimine is an orientation which maximises overlap between the pi orbitals of the conjugated system, stabilising the transition state and therefore accelerating the rate of bond cleavage in the decarboxylation step. steric role, transition state 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

References

  1. Taylor JL et al. (2015), Biochim Biophys Acta, 1854, 146-155. Directed evolution of the substrate specificity of dialkylglycine decarboxylase. DOI:10.1016/j.bbapap.2014.12.003. PMID:25500286.
  2. Fogle EJ et al. (2010), Biochemistry, 49, 6485-6493. Mutational Analysis of Substrate Interactions with the Active Site of Dialkylglycine Decarboxylase. DOI:10.1021/bi100648w. PMID:20540501.
  3. Fogle EJ et al. (2005), Biochemistry, 44, 16392-16404. Role of Q52 in Catalysis of Decarboxylation and Transamination in Dialkylglycine Decarboxylase†. DOI:10.1021/bi051475b. PMID:16342932.
  4. Zhou X et al. (2001), Biochemistry, 40, 1367-1377. Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase†. DOI:10.1021/bi001237a.
  5. Zhou X et al. (1999), Biochemistry, 38, 311-320. pH Studies on the Mechanism of the Pyridoxal Phosphate-Dependent Dialkylglycine Decarboxylase†. DOI:10.1021/bi981455s. PMID:9890912.
  6. Malashkevich VN et al. (1999), J Mol Biol, 294, 193-200. Crystal structures of dialkylglycine decarboxylase inhibitor complexes. DOI:10.1006/jmbi.1999.3254. PMID:10556038.
  7. Toney MD et al. (1995), J Mol Biol, 245, 151-179. Structural and Mechanistic Analysis of Two Refined Crystal Structures of the Pyridoxal Phosphate-dependent Enzyme Dialkylglycine Decarboxylase. DOI:10.1006/jmbi.1994.0014. PMID:7799433.
  8. Toney MD et al. (1993), Science, 261, 756-759. Dialkylglycine decarboxylase structure: bifunctional active site and alkali metal sites. PMID:8342040.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys272A covalent catalysis, proton shuttle (general acid/base)
Glu210A steric role
Arg406A steric role
Glu210A transition state stabiliser
Arg406A transition state stabiliser
Trp138A steric role
Asp243A electrostatic stabiliser, increase electrophilicity
Gln246A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

James W. Murray, Craig Porter