D-alanine transaminase

 

D-Amino acid aminotransferase (D-aAt) catalyses the transamination of various D-amino acids, forming their respective keto acids. The enzyme has no sequence similarity to the well studied L-amino acid aminotransferase but does have significant sequence overlap with a bacterial branched-chain L-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase. D-aAt is essential for the synthesis of bacterial cell wall components and has been a target of research in the development of antimicrobial agents.

Acts on the D-isomers of alanine, leucine, aspartate, glutamate, aminobutyrate, norvaline and asparagine. The enzyme transfers an amino group from a substrate D-amino acid to the pyridoxal phosphate cofactor to form pyridoxamine and an alpha-keto acid in the first half-reaction. The second-half reaction is the reverse of the first, transferring the amino group from the pyridoxamine to a second alpha-keto acid to form the product D-amino acid via a ping-pong mechanism. This is an important process in the formation of D-alanine and D-glutamate, which are essential bacterial cell wall components.

 

Reference Protein and Structure

Sequence
P19938 UniProt (2.6.1.21) IPR005784 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus sp. YM-1 (Bacteria) Uniprot
PDB
1daa - CRYSTALLOGRAPHIC STRUCTURE OF D-AMINO ACID AMINOTRANSFERASE COMPLEXED WITH PYRIDOXAL-5'-PHOSPHATE (1.94 Å) PDBe PDBsum 1daa
Catalytic CATH Domains
3.20.10.10 CATHdb 3.30.470.10 CATHdb (see all for 1daa)
Cofactors
Pyridoxal 5'-phosphate(2-) (1), Water (1)
Click To Show Structure

Enzyme Reaction (EC:2.6.1.21)

D-glutamate(1-)
CHEBI:29986ChEBI
+
pyruvate
CHEBI:15361ChEBI
→
2-oxoglutarate(2-)
CHEBI:16810ChEBI
+
D-alanine zwitterion
CHEBI:57416ChEBI
Alternative enzyme names: D-alanine aminotransferase, D-aspartate transaminase, D-aspartic aminotransferase, D-amino acid transaminase, D-amino acid aminotransferase, D-aspartate aminotransferase, D-alanine-D-glutamate transaminase, D-alanine transaminase,

Enzyme Mechanism

Introduction

Upon binding of a substrate amino acid, a transaldimation reaction occurs, releasing Lys145 from the internal aldimine and forming an external aldimine between the substrate and PLP cofactor. Lys145 then acts as the general base in the next step, driving the 1,3 prototropic shift that converts the internal aldimine into a ketimine intermediate. Next, this ketimine is hydrolysed to form pyridoxamine phosphate and an alpha-keto acid. The second half of the reaction is the reversal of these steps with a different keto acid.

Catalytic Residues Roles

UniProt PDB* (1daa)
Lys146 Lys145A The residue forms a covalent Schiff base link to the PLP cofactor. Once the residue has been displaced during the formation of the external aldimine, the basic side group is free to act as a general base, driving the 1,3 prototropic shift. This converts the internal aldimine to the ketimine intermediate. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge, electron pair acceptor, electron pair donor
Tyr32 Tyr31A The residue's phenolic oxygen hydrogen bonds to the catalytic base Lys145. This interaction is thought to activate Lys145 towards its function as a general base. Mutagenesis has also implicated the residue in maintaining stereochemical fidelity. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Leu202 Leu201A Acts to hold the PLP cofactor in place such that the reaction occurs with the correct sterochemistry. van der waals interaction, steric role
Glu178 Glu177A The residue's negatively charged side chain hydrogen bonds to the nitrogen of the pyridinium ring , an interaction which is thought to stabilise the carbanion intermediates of the reaction. activator, hydrogen bond acceptor, electrostatic 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

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

References

  1. Peisach D et al. (1998), Biochemistry, 37, 4958-4967. Crystallographic Study of Steps along the Reaction Pathway ofd-Amino Acid Aminotransferase†,‡. DOI:10.1021/bi972884d. PMID:9538014.
  2. van Ophem PW et al. (1999), Biochemistry, 38, 1323-1331. Effects of the E177K Mutation ind-Amino Acid Transaminase. Studies on an Essential Coenzyme Anchoring Group That Contributes to Stereochemical Fidelity†,‡. DOI:10.1021/bi982414z. PMID:9930994.
  3. Sugio S et al. (1998), Protein Eng, 11, 613-619. Crystal structures of L201A mutant of D-amino acid aminotransferase at 2.0 A resolution: implication of the structural role of Leu201 in transamination. DOI:10.1093/protein/11.8.613. PMID:9749913.
  4. Sugio S et al. (1995), Biochemistry, 34, 9661-9669. Crystal Structure of a D-Amino Acid Aminotransferase: How the Protein Controls Stereoselectivity. DOI:10.1021/bi00030a002. PMID:7626635.
  5. Tanizawa K et al. (1989), J Biol Chem, 264, 2445-2449. Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination. PMID:2914916.

Step 1. The amine of the substrate L-glutamate attacks the PLP cofactor in a nucleophilic addition and the bound Lys145 deprotonates the newly attached amine.

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Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, electrostatic stabiliser
Lys145A covalently attached, hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction
Lys145A proton acceptor, electron pair acceptor

Chemical Components

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

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction
Lys145A nucleofuge

Chemical Components

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

Step 3. Lys145 deprotonates the CH adjacent to the bound amine, resulting in double bond rearrangement as the PLP acts as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond acceptor, hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction
Lys145A 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 deprotonates Lys145.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A hydrogen bond acceptor, activator
Leu201A steric role, van der waals interaction
Lys145A proton donor

Chemical Components

proton transfer, intermediate formation

Step 5. Lys145 deprotonates water, which initiates a nucleophilic attack on the carbon of the C=N group in an addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond acceptor, hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction
Lys145A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation

Step 6. The secondary amine deprotonates the attached hydroxyl group, initiating an elimination which releases 2-oxoglutarate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction

Chemical Components

ingold: intramolecular elimination, overall product formed, intermediate collapse, intermediate formation

Step 7. The amine of PMP initiates a nucleophilic attack on the carbonyl carbon of pyruvate. The oxyanion deprotonates the newly formed secondary amine in the first step of a Schiff base formation.

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Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction

Chemical Components

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

Step 8. The secondary amine initiates an elimination, forming the Schiff base and releasing water with concomitant deprotonation of Lys145.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction
Lys145A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, intermediate formation, dehydration, schiff base formed

Step 9. Lys145 deprotonates the CH2 adjacent to the nitrogen, resulting in double bond rearrangement as the PLP acts as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond acceptor, hydrogen bond donor
Glu177A electrostatic stabiliser, hydrogen bond acceptor
Leu201A steric role, van der waals interaction
Lys145A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 10. The PLP feeds the electrons back, the N+=C bond deprotonates Lys145.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A hydrogen bond acceptor, activator
Leu201A steric role, van der waals interaction
Lys145A proton donor

Chemical Components

proton transfer, intermediate formation

Step 11. The amine of Lys145 attacks the PLP in a nucleophilic addition reaction, the secondary amine of the attached substrate reprotonates from the bound Lys145.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Lys145A hydrogen bond donor
Glu177A hydrogen bond acceptor, electrostatic stabiliser
Leu201A steric role, van der waals interaction
Lys145A nucleophile, proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation

Step 12. The secondary amine that results from the initial attack initiates an elimination of the covalently bound product, resulting in alanine and the regenerated PLP cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr31A hydrogen bond donor, electrostatic stabiliser
Lys145A covalently attached, hydrogen bond donor
Glu177A hydrogen bond acceptor, electrostatic stabiliser
Leu201A steric role, van der waals interaction
Lys145A electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, overall product formed, intermediate collapse, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The amine of the substrate L-glutamate attacks the PLP cofactor in a nucleophilic addition and the bound Lys145 deprotonates the newly attached amine.

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. Lys145 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 deprotonates Lys145.

Step 5. Lys145 deprotonates water, which initiates a nucleophilic attack on the carbon of the C=N group in an addition reaction.

Step 6. The secondary amine deprotonates the attached hydroxyl group, initiating an elimination which releases 2-oxoglutarate.

Step 7. The amine of PMP initiates a nucleophilic attack on the carbonyl carbon of pyruvate. The oxyanion deprotonates the newly formed secondary amine in the first step of a Schiff base formation.

Step 8. The secondary amine initiates an elimination, forming the Schiff base and releasing water with concomitant deprotonation of Lys145.

Step 9. Lys145 deprotonates the CH2 adjacent to the nitrogen, resulting in double bond rearrangement as the PLP acts as an electron sink.

Step 10. The PLP feeds the electrons back, the N+=C bond deprotonates Lys145.

Step 11. The amine of Lys145 attacks the PLP in a nucleophilic addition reaction, the secondary amine of the attached substrate reprotonates from the bound Lys145.

Step 12. The secondary amine that results from the initial attack initiates an elimination of the covalently bound product, resulting in alanine and the regenerated PLP cofactor.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, James W. Murray, Craig Porter