Structure–function analyses of the G729R 2-oxoadipate dehydrogenase genetic variant associated with a disorder of l-lysine metabolism

Abstract

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in l-lysine catabolism. Genetic findings have linked mutations in the DHTKD1 gene to several metabolic disorders. These include α-aminoadipic and α-ketoadipic aciduria (AMOXAD), a rare disorder of l-lysine, l-hydroxylysine, and l-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments. A heterozygous missense mutation, c.2185G→A (p.G729R), in DHTKD1 has been identified in most AMOXAD cases. Here, we report that the G729R E1a variant when assembled into OADHc in vitro displays a 50-fold decrease in catalytic efficiency for NADH production and a significantly reduced rate of glutaryl-CoA production by dihydrolipoamide succinyl-transferase (E2o). However, the G729R E1a substitution did not affect any of the three side-reactions associated solely with G729R E1a, prompting us to determine the structure–function effects of this mutation. A multipronged systematic analysis of the reaction rates in the OADHc pathway, supplemented with results from chemical cross-linking and hydrogen–deuterium exchange MS, revealed that the c.2185G→A DHTKD1 mutation affects E1a–E2o assembly, leading to impaired channeling of OADHc intermediates. Cross-linking between the C-terminal region of both E1a and G729R E1a with the E2o lipoyl and core domains suggested that correct positioning of the C-terminal E1a region is essential for the intermediate channeling. These findings may inform the development of interventions to counter the effects of pathogenic DHTKD1 mutations.

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

“…In comparison, the value of K d of 0.041 μM (for hE2o 1-173 di-domain) and K d of 0.060 μM (for E2o 144-386 core domain) were reported for hE1o (24), suggesting a different binding mode of hE1o and hE1a with the same hE2o component. Also in HDX-MS studies, it was demonstrated that the region from the N-terminal end of hE1a comprising residues 24-47 experienced significant protection from deuterium uptake in the sub-complex with hE2o, similarly to that observed for hE1o above (residues 27-40) (25). Importantly, two regions of hE1a comprising residues 485-518, which according to the recently reported X-ray structure (22) belong to the linker region connecting two halves of the hE1a monomer, and peptides in the C-terminal region of hE1a comprising residues 589-609, 646-664, and 847-874 experienced protection from deuterium uptake on interaction with hE2o (25).…”

“…Also in HDX-MS studies, it was demonstrated that the region from the N-terminal end of hE1a comprising residues 24-47 experienced significant protection from deuterium uptake in the sub-complex with hE2o, similarly to that observed for hE1o above (residues 27-40) (25). Importantly, two regions of hE1a comprising residues 485-518, which according to the recently reported X-ray structure (22) belong to the linker region connecting two halves of the hE1a monomer, and peptides in the C-terminal region of hE1a comprising residues 589-609, 646-664, and 847-874 experienced protection from deuterium uptake on interaction with hE2o (25). To probe deep inside the assembly of hE1o and hE1a to E2o, we next turned to chemical cross-linking mass spectrometry (CL-MS) to identify loci of interaction in binary E1-E2 sub-complexes.…”

“…(ii) An initial formation of the hE1o-hE2o interaction facilitates assembly with hE3 to form the hOGDHc (24). In studies with hE1a by using the fluorescent labeled E2o proteins, it was demonstrated that hE1a binding to hE2o is strong enough with values of K d =1.12 μM for pyrene-labeled E2o 1-173 di-domain, comprising lipoyl domain and linker region, and K d =3.98 μM for the pyrene-labeled E2o 144-386 core domain (25). In comparison, the value of K d of 0.041 μM (for hE2o 1-173 di-domain) and K d of 0.060 μM (for E2o 144-386 core domain) were reported for hE1o (24), suggesting a different binding mode of hE1o and hE1a with the same hE2o component.…”

“…On the hE1a, the inter-component cross-links were identified for Lys residues from the N-terminal region (Lys 37 , Lys 148 , and Lys 188 ), residues in the ThDP and Mg 2+ -binding region (Lys 300 and Lys 450 ) and those located in the C-terminal region (Lys 818 , Lys 826 /Lys 827 , and Lys 852 /Lys 854 ) (25). On hE2o, the cross-linked Lys 24 , Lys 43 , Lys 66 , Lys 78 , and Lys 87 are from the lipoyl domain; Lys 150 and Lys 159 are from the linker region of hE2o; and Lys 240 , Lys 286 , Lys 289 , Lys 342 , Lys 371 , and Lys 373 are from the hE2o core domain (25).…”

“…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). Experiments have revealed two important facts about cross-talk between the human OGDHc (hOGDHc) in TCA cycle and human E1a (hE1a) in the L-lysine metabolic pathway: (a) The hE1o and hE1a share the same E2o and E3 components for their assembly into the corresponding OGDHc and 2-oxoadipate dehydrogenase complex (OADHc) to perform their function in two distinct metabolic pathways (10).…”