A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

J Bacteriol. 2017 Oct 17;199(22):e00342-17. doi: 10.1128/JB.00342-17. Print 2017 Nov 15.


Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10 Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH.IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.

Keywords: NAD-independent d-lactate dehydrogenase; Pseudomonas putida; electron transfer; flavoprotein; iron-sulfur protein; lactate utilization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Coenzymes
  • Cytochromes c / metabolism
  • Electrons
  • Flavin-Adenine Dinucleotide / metabolism*
  • Iron-Sulfur Proteins / metabolism*
  • Lactate Dehydrogenases / genetics*
  • Lactate Dehydrogenases / isolation & purification
  • Lactate Dehydrogenases / metabolism*
  • Lactic Acid / metabolism*
  • Mutagenesis, Site-Directed
  • NAD / metabolism
  • Oxidation-Reduction
  • Pseudomonas putida / enzymology
  • Quinones / metabolism*
  • Ubiquinone / analogs & derivatives
  • Ubiquinone / metabolism


  • Bacterial Proteins
  • Coenzymes
  • Iron-Sulfur Proteins
  • Quinones
  • NAD
  • Ubiquinone
  • Flavin-Adenine Dinucleotide
  • Lactic Acid
  • Cytochromes c
  • Lactate Dehydrogenases
  • D-lactate dehydrogenase
  • coenzyme Q10