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

Abstract

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 FADcontaining 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.