D-lactate dehydrogenase

 

D -Lactate dehydrogenase from Lactobacillus bulgaricus, a homodimer with 332 amino acid, acts at the last step of the glycolytic pathway under anaerobic conditions, allowing re-oxidation of NAD, which is necessary for glycolysis. The enzyme catalyses the NAD-dependent conversion of pyruvate into the D -isomer of lactic acid. The reaction is reversible: pyruvate reduction (the forward reaction) shows a maximum rate at pH 7.5 and D-lactate oxidation (the inverse reaction) at pH 8.0. The reaction leading to the other enantiomer, L-lactic acid, is catalysed by another enzyme, L-lactate dehydrogenase (L-LDH). While L-LDH has a wide occurrence in nature, D-LDH is found only in invertebrates, lower fungi and prokaryotic organisms. Lactic bacteria possess either one or both enzymes. In Lactobacillus bulgaricus, commonly used in the dairy industry for the production of yoghurt, more than 90% of the pyruvate is converted into D-lactate.

 

Reference Protein and Structure

Sequence
P26297 UniProt (1.1.1.28) IPR006139 (Sequence Homologues) (PDB Homologues)
Biological species
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (Bacteria) Uniprot
PDB
1j49 - INSIGHTS INTO DOMAIN CLOSURE, SUBSTRATE SPECIFICITY AND CATALYSIS OF D-LACTATE DEHYDROGENASE FROM LACTOBACILLUS BULGARICUS (2.2 Å) PDBe PDBsum 1j49
Catalytic CATH Domains
3.40.50.720 CATHdb (see all for 1j49)
Click To Show Structure

Enzyme Reaction (EC:1.1.1.28)

NAD(1-)
CHEBI:57540ChEBI
+
(R)-lactate
CHEBI:16004ChEBI
→
pyruvate
CHEBI:15361ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
Alternative enzyme names: D-(-)-lactate dehydrogenase (NAD), D-lactic acid dehydrogenase, D-lactic dehydrogenase, D-specific lactic dehydrogenase, Lactic acid dehydrogenase,

Enzyme Mechanism

Introduction

With reference to the known mechanism of L-lactate dehydrogenases, the reaction proceeds first with a proton being donated from (R)-lactate by His297 then a simultaneous proton donation to His297 and a hydride being transferred to NAD+, with help by Arg236 and Asp260 residues.

Catalytic Residues Roles

UniProt PDB* (1j49)
Asp260 Asp260A Asp260 twists the NAD+ carboxamide by 21 degrees from the pyridine plane and fixing its oxygen in the cis-orientation with respect to C4N. This conformation has been shown to be unfavourable in isolated NADH/NAD+ molecules, because of the partial double-bond character of the C3N±C4N bond, but it is important in the activation of hydride transfer in dehydrogenases. steric role
Glu265 Glu265A H-bonds to His297 to stabilise the protonated form, increasing its pKa and aid in its function as a base in the concerted step electrostatic stabiliser
His297 His297A Acts as a general acid/base and abstracts and donates a proton from pyruvate to form D-lactate. proton acceptor, proton donor
Arg236 Arg236A Polarises the carbonyl bond in the pyruvate molecule to activate the hydride transfer. 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

proton transfer, hydride transfer, aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed

References

  1. Razeto A et al. (2002), J Mol Biol, 318, 109-119. Domain Closure, Substrate Specificity and Catalysis of d-Lactate Dehydrogenase from Lactobacillus bulgaricus. DOI:10.1016/s0022-2836(02)00086-4. PMID:12054772.
  2. Kochhar S et al. (2000), Eur J Biochem, 267, 1633-1639. Roles of His205, His296, His303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase. DOI:10.1046/j.1432-1327.2000.01155.x. PMID:10712593.

Step 1. His297 abstracts a proton from the hydroxyl group of D-lactate. There is aromatic bimolecular nucleophilic addition of a hydride from the reactive carbon of D-lactate to NAD+, forming NADH and the product. Glu265 stabilises the protonated form of His297 and increases its pKa.

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Catalytic Residues Roles

Residue Roles
Arg236A electrostatic stabiliser
Asp260A steric role
Glu265A electrostatic stabiliser
His297A proton acceptor

Chemical Components

proton transfer, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, overall product formed

Step 2. In an inferred reaction step His297 is deprotonated to regenerate the native state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His297A proton donor

Chemical Components

proton transfer
Download: Image, Marvin File

Step 1. His297 abstracts a proton from the hydroxyl group of D-lactate. There is aromatic bimolecular nucleophilic addition of a hydride from the reactive carbon of D-lactate to NAD+, forming NADH and the product. Glu265 stabilises the protonated form of His297 and increases its pKa.

Step 2. In an inferred reaction step His297 is deprotonated to regenerate the native state of the enzyme.

Products.

Contributors

Anna Waters, Craig Porter, Gemma L. Holliday, Amelia Brasnett