Understanding of the 2-oxoglutarate dehydrogenase and 2-oxoadipate dehydrogenase assembly with the E2o core relevant to a hybrid complex formation

The 2-oxoglutarate (OG) dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid cycle (TCA) and comprises multiple copies of three components: 2-oxoglutarate dehydrogenase (hE1o), dihydrolipoamide succinyltransferase (hE2o), and dihydrolipoamide dehydrogenase (hE3). The OGDHc is one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels and impacts cell metabolic and cell signaling pathways through the coupling of OG metabolism to gene transcription, related to tumor cell proliferation and aging. The reduced OGDHc activity is linked to a number of neurodegenerative diseases. Evidence was obtained for the formation of a hybrid 2-oxo acid dehydrogenase complex between the OGDHc and its homologue 2-oxoadipate (OA) dehydrogenase (hE1a) in the L-lysine metabolic pathway, suggesting a potential cross-talk between the two distinct metabolic pathways. These findings raised fundamental questions about assembly of hE1a and hE1o to the hE2o core. Due to the lack of an atomic structure of the OGDHc from any sources, and of knowledge about exact distribution of components around the E2 core, hydrogen/deuterium exchange (HDX-MS) and chemical cross-linking mass spectrometry (CL-MS) have been carried out in binary hE1o-hE2o, hE1a-hE2o, hE1o-hE3 and hE2o-hE3 sub-complexes followed by structural modeling. Here we report findings that revealed some similarities in the assembly of hE1o and hE1a to the hE2o core. At the same time, three regions of the hE2o core comprising residues 191-208, 273-288, and 370-386 revealed a different binding mode to hE1o and hE1a, suggesting that hE2o can differentiate between these two proteins that may have physiological consequences.

Crystal structure and interaction studies of human DHTKD1 provide insight into a mitochondrial megacomplex in lysine catabolism

DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid dehydrogenases. In complex with E2 (dihydrolipoamide succinyltransferase, DLST) and E3 (dihydrolipoamide dehydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative decarboxylation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Å resolution crystal structure of human DHTKD1 is solved in complex with the thiamine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) dehydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Å resolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-aminoadipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.

Structure of the dihydrolipoamide succinyltransferase catalytic domain from Escherichia coli in a novel crystal form: a tale of a common protein crystallization contaminant

The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.7 Å) were obtained and diffracted to 3.0 Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) program, it was discovered that the structure corresponded to dihydrolipoamide succinyltransferase from Escherichia coli (PDB entry 1c4t), which is considered to be a common crystallization contaminant protein. The structure was refined to an R work of 23.0% and an R free of 27.2% at 3.0 Å resolution. The structure was compared with others of the same protein deposited in the PDB. This is the first report of the structure of dihydrolipoamide succinyltransferase isolated without an expression tag and in this novel crystal form.