Abstract
Mitochondrial L-2-hydroxyglutarate dehydrogenase (L2HGDH) is a FAD-containing membrane protein that oxidizes L-2-hydroxyglutarate (L-2-HG) to 2-oxoglutarate (2-OG). L2HGDH dysfunction is associated with human diseases, such as neurometabolic disorders and cancer. Recently, the first crystal structure of Drosophila melanogaster L2HGDH (DmL2HGDH) bound with FAD and 2-OG was reported. Structural analysis, together with mutagenesis and activity measurements, revealed the oxidation mechanism of L-2-HG. However, the physiologically relevant electron acceptor has not yet been elucidated. While bacterial L2HGDH has been proposed to reduce ubiquinone, this remains controversial. To understand the full reaction mechanism of L2HGDH, identification of the electron acceptor and characterization of its interaction with the enzyme are necessary. In this study, we demonstrate that DmL2HGDH transfers electrons from L-2-HG to decylubiquinone, a ubiquinone analog that is sparingly soluble in aqueous buffers and used for in vitro studies. The reaction was moderately inhibited by ferulenol, which inhibits some quinone reductases, including the homologous enzyme malate-quinone oxidoreductase (MQO). Interestingly, the observed kinetic behavior differs from that of MQO. Using AlphaFold3 and molecular dynamics, we provide insights into the interaction between DmL2HGDH and ubiquinone and propose a mechanism for ubiquinone reduction.
