Alanine dehydrogenase

 

Alanine dehydrogenase catalyses the NADH-dependent reversible reductive amination of pyruvate to L-alanine. The enzyme plays an important role in the carbon and nitrogen metabolism of micro-organisms and is a key factor in the assimilation of L-alanine as an energy source through the tricarboxylic acid cycle during sporulation.

 

Reference Protein and Structure

Sequence
O52942 UniProt (1.4.1.1) IPR008141 (Sequence Homologues) (PDB Homologues)
Biological species
Phormidium lapideum (Bacteria) Uniprot
PDB
1pjb - L-ALANINE DEHYDROGENASE (2.1 Å) PDBe PDBsum 1pjb
Catalytic CATH Domains
3.40.50.720 CATHdb (see all for 1pjb)
Click To Show Structure

Enzyme Reaction (EC:1.4.1.1)

NAD(1-)
CHEBI:57540ChEBI
+
water
CHEBI:15377ChEBI
+
L-alanine zwitterion
CHEBI:57972ChEBI
→
NADH(2-)
CHEBI:57945ChEBI
+
pyruvate
CHEBI:15361ChEBI
+
ammonium
CHEBI:28938ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Alpha-alanine dehydrogenase, L-alanine dehydrogenase, AlaDH, NAD-dependent alanine dehydrogenase, NAD-linked alanine dehydrogenase, NADH-dependent alanine dehydrogenase, Alanine oxidoreductase,

Enzyme Mechanism

Introduction

Biochemical data suggests that the mechanism proceeds through iminopyruvate and carbinolamine intermediates. The scheme proposes that the substrate carboxyl group is bound by both Lys74 and Arg15. These polarise the carbonyl in order to activate the substrate. The first step of the reaction in the direction of oxidative deamination involves hydride transfer between the C-alpha of alanine and the nicotinamide ring to produce a protonated iminopyruvate. Subsequently a conformational change occurs that allows a water molecule to enter the active site attacking the iminopyruvate to produce a carbinolamine in a process thought to be facilitated by hydrogen bonding of the water to a cationic acid, His95. The activity of His95 as part of a proton shuffle is facilitated by the close association of two acidic residues, Glu117 and Asp269. Finally the carbinolamine collapses to yield pyruvate and ammonia to complete the proposed reaction.

Catalytic Residues Roles

UniProt PDB* (1pjb)
Tyr93 Tyr93A Acts as a steric inhibitor which affects the stereochemistry of the mechanism, however, the function of this residue is only tentatively assigned. steric role, polar/non-polar interaction
His95, Glu117 His95A, Glu117A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Lys74, Arg15 Lys74A, Arg15A The substrate carboxyl group is bound by both Lys74 and Arg15. These polarise the carbonyl in order to activate the substrate. activator, hydrogen bond donor, electrostatic stabiliser
Asp269 Asp269A Asp269 may replace Glu117 when structural changes occur in the enzyme. Acts as a general acid/base proton acceptor, proton 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

hydride transfer, unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation, proton transfer, bimolecular nucleophilic addition, proton relay, intramolecular elimination, deamination, intermediate collapse, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Baker PJ et al. (1998), Nat Struct Biol, 5, 561-567. Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase. DOI:10.1038/817. PMID:9665169.
  2. Lerchner A et al. (2016), Biotechnol Appl Biochem, 63, 616-624. Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity. DOI:10.1002/bab.1414. PMID:26202482.
  3. Li XY et al. (2015), Int J Mol Sci, 16, 29383-29397. Domain Motions and Functionally-Key Residues of l-Alanine Dehydrogenase Revealed by an Elastic Network Model. DOI:10.3390/ijms161226170. PMID:26690143.
  4. Ling B et al. (2014), J Mol Graph Model, 50, 61-70. Molecular dynamics simulations of mutated Mycobacterium tuberculosis l-alanine dehydrogenase to illuminate the role of key residues. DOI:10.1016/j.jmgm.2014.03.008. PMID:24763245.
  5. Ling B et al. (2012), J Mol Graph Model, 35, 1-10. Molecular dynamics simulations of the coenzyme induced conformational changes of Mycobacterium tuberculosis l-alanine dehydrogenase. DOI:10.1016/j.jmgm.2012.01.005. PMID:22459692.
  6. Agren D et al. (2008), J Mol Biol, 377, 1161-1173. Three-Dimensional Structures of Apo- and Holo-l-Alanine Dehydrogenase from Mycobacterium tuberculosis Reveal Conformational Changes upon Coenzyme Binding. DOI:10.1016/j.jmb.2008.01.091. PMID:18304579.
  7. Tripathi SM et al. (2008), Proteins, 72, 1089-1095. Crystal structures of the Mycobacterium tuberculosis secretory antigen alanine dehydrogenase (Rv2780) in apo and ternary complex forms captures “open” and “closed” enzyme conformations. DOI:10.1002/prot.22101. PMID:18491387.

Step 1. pH profile studies suggest that the alanine binds as the monoanion. The amine of alanine initiates the elimination of the hydride ion, which adds to NAD. Subsequently, a conformational change occurs that allows a water molecule to enter the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A hydrogen bond donor, electrostatic stabiliser
Tyr93A polar/non-polar interaction
Glu117A hydrogen bond acceptor
His95A hydrogen bond acceptor, hydrogen bond donor
Arg15A activator

Chemical Components

hydride transfer, ingold: unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed, intermediate formation

Step 2. Asp269 deprotonates His95, which undergoes double bond rearrangement and deprotonates water, which initiates a nucleophilic attack on the carbinolamine carbon in an addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A hydrogen bond donor
Glu117A hydrogen bond acceptor
His95A proton relay, hydrogen bond acceptor, hydrogen bond donor
Tyr93A steric role, polar/non-polar interaction
Glu117A electrostatic stabiliser
Arg15A activator
His95A proton acceptor
Asp269A proton acceptor
His95A proton donor

Chemical Components

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

Step 3. The amine deprotonates the hydroxide added, which initiates an elimination of ammoina.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A hydrogen bond donor, electrostatic stabiliser, activator
His95A hydrogen bond acceptor, hydrogen bond donor
Tyr93A steric role, polar/non-polar interaction
Glu117A hydrogen bond donor
Arg15A activator

Chemical Components

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

Step 4. Ammonia deprotonates His95, which undergoes double bond rearrangement and deprotonates Asp269 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His95A hydrogen bond acceptor, hydrogen bond donor, proton relay
Glu117A hydrogen bond donor, electrostatic stabiliser
Asp269A proton donor
His95A proton donor, proton acceptor

Chemical Components

proton transfer, overall product formed, intermediate terminated, native state of enzyme regenerated, proton relay, inferred reaction step
Download: Image, Marvin File

Step 1. pH profile studies suggest that the alanine binds as the monoanion. The amine of alanine initiates the elimination of the hydride ion, which adds to NAD. Subsequently, a conformational change occurs that allows a water molecule to enter the active site.

Step 2. Asp269 deprotonates His95, which undergoes double bond rearrangement and deprotonates water, which initiates a nucleophilic attack on the carbinolamine carbon in an addition reaction.

Step 3. The amine deprotonates the hydroxide added, which initiates an elimination of ammoina.

Step 4. Ammonia deprotonates His95, which undergoes double bond rearrangement and deprotonates Asp269 in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Alex Gutteridge, Craig Porter