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

Abstract

<jats:p>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.</jats:p>

Selective Inhibition of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases by the Phosphonate Analogs of Their 2-Oxo Acid Substrates

“…To assess how the phosphonates interact with the active site of OADH, the recently published structure of human OADH was used [PDB ID: 6SY1 (Bezerra et al, 2020 )]. The human enzyme has 89% sequence identity with the mature rat protein, i.e., the protein without the N-terminal mitochondrial localization signal, absent in the crystal structure ( Figure 8 ).…”

“…Structure of rat OGDH was modeled using SWISSModel web-server (Waterhouse et al, 2018 ), with OGDH from M. smegmatis in complex with the post-decarboxylation ThDP adduct [PDB ID: 2Y0P, (Wagner et al, 2011 )] serving as a template. The modeled structure was superimposed onto that of human DHTKD1 [PDB ID: 6SY1, (Bezerra et al, 2020 )]. Each of the structures was then aligned to M. smegmatis OGDH in complex with SP * ThDP (PDB ID: 6R29, [Wagner et al, 2019 )].…”

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

“…There are, however, X-ray structures available for the N-terminally truncated E. coli E1o Δ77 (20) and the N-terminally truncated Mycobacterium smegmatis α-ketoglutarate decarboxylase ( Ms KGD Δ115 ) (21). Recently, X-ray structures were reported for the N-terminally truncated hE1a at 1.9Å (22) and at 2.25Å (23) resolution. Due to the lack of an atomic structure of the OGDHc from any sources and to the lack of knowledge about exact distribution of components around the E2 core due to their structural dynamics, hydrogen/deuterium exchange (HDX-MS) and chemical cross-linking (CL-MS) mass spectrometry have been carried out in binary hE1o-hE2o, hE1a-hE2o, hE1o-hE3 and hE2o-hE3 sub-complexes in by the Jordan group (24,25).…”

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“…Structure of rat OGDH was modeled using SWISSModel web-server (Waterhouse et al, 2018), with OGDH from M. smegmatis in complex with the post-decarboxylation ThDP adduct [PDB ID: 2Y0P, (Wagner et al, 2011)] serving as a template. The modeled structure was superimposed onto that of human DHTKD1 [PDB ID: 6SY1, (Bezerra et al, 2020)]. Each of the structures was then aligned to M. smegmatis OGDH in complex with SP * ThDP (PDB ID: 6R29, [Wagner et al, 2019)].…”

“…To assess how the phosphonates interact with the active site of OADH, the recently published structure of human OADH was used [PDB ID: 6SY1 (Bezerra et al, 2020)]. The human enzyme has 89% sequence identity with the mature rat protein, i.e., the protein without the N-terminal mitochondrial localization signal, absent in the crystal structure (Figure 8).…”