The mitochondrial 2-oxoadipate and 2-oxoglutarate dehydrogenase complexes share their E2 and E3 components for their function and both generate reactive oxygen species.

Herein are reported unique properties of the novel human thiamin diphosphate (ThDP)-dependent enzyme 2-oxoadipate dehydrogenase (hE1a), known as dehydrogenase E1 and transketolase domain-containing protein 1 that is encoded by the DHTKD1 gene. It is involved in the oxidative decarboxylation of 2-oxoadipate (OA) to glutaryl-CoA on the final degradative pathway of L-lysine and is critical for mitochondrial metabolism. Functionally active recombinant hE1a has been produced according to both kinetic and spectroscopic criteria in our toolbox leading to the following conclusions: (i) The hE1a has recruited the dihydrolipoyl succinyltransferase (hE2o) and the dihydrolipoyl dehydrogenase (hE3) components of the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex (OGDHc) for its activity. (ii) 2-Oxoglutarate (OG) and 2-oxoadipate (OA) could be oxidized by hE1a, however, hE1a displays an approximately 49-fold preference in catalytic efficiency for OA over OG, indicating that hE1a is specific to the 2-oxoadipate dehydrogenase complex. (iii) The hE1a forms the ThDP-enamine radical from OA according to electron paramagnetic resonance detection in the oxidative half reaction, and could produce superoxide and H2O2 from decarboxylation of OA in the forward physiological direction, as also seen with the 2-oxoglutarate dehydrogenase hE1o component. (iv) Once assembled to complex with the same hE2o and hE3 components, the hE1o and hE1a display strikingly different regulation: both succinyl-CoA and glutaryl-CoA significantly reduced the hE1o activity, but not the activity of hE1a.

The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate.

Recently, we reported that the human 2-oxoglutarate dehydrogenase (hE1o) component of the 2-oxoglutarate dehydrogenase complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18]]. We here report that hE1o can also utilize the next higher homologue of OG, 2-oxoadipate (OA) as a substrate according to multiple criteria in our toolbox: (i) Both E1o-specific and overall complex activities (NADH production) were detected using OA as a substrate; (ii) Two post-decarboxylation intermediates were formed by hE1o from OA, the ThDP-enamine and the C2α-hydroxyalkyl-ThDP, with nearly identical rates for OG and OA; (iii) Both OG and OA could reductively acylate lipoyl domain created from dihydrolipoyl succinyltransferase (E2o); (iv) Both OG and OA gave α-ketol carboligaton products with glyoxylate, but with opposite chirality; a finding that could be of utility in chiral synthesis; (v) Dioxygen could oxidize the ThDP-derived enamine from both OG and OA, leading to ThDP-enamine radical and generation of superoxide and H2O2. While the observed oxidation-reduction with dioxygen is only a side reaction of the predominant physiological product glutaryl-CoA, the efficiency of superoxide/ H2O2 production was 7-times larger from OA than from OG, making the reaction of OGDHc with OA one of the important superoxide/ H2O2 producers among 2-oxo acid dehydrogenase complexes in mitochondria.