An Updated View on the Molecular Pathomechanisms of Human Dihydrolipoamide Dehydrogenase Deficiency in Light of Novel Crystallographic Evidence
- 140 Downloads
Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early death; the highest carrier rate has been found among Ashkenazi Jews. Acute clinical episodes usually involve severe metabolic decompensation and lactate acidosis that result in neurological, cardiological, and/or hepatological manifestations. Clinical severity is due to the fact that LADH is a common E3 subunit to the alpha-ketoglutarate, pyruvate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes, and is also a constituent in the glycine cleavage system, thus a loss in LADH function adversely affects multiple key metabolic routes. However, the severe clinical pictures frequently still do not parallel the LADH activity loss, which implies the involvement of auxiliary biochemical mechanisms; enhanced reactive oxygen species generation as well as affinity loss for multienzyme complexes proved to be key auxiliary exacerbating pathomechanisms. This review provides an overview and an up-to-date molecular insight into the pathomechanisms of this disease in light of the structural conclusions drawn from the first crystal structure of a disease-causing hE3 variant determined recently in our laboratory.
KeywordsDihydrolipoamide dehydrogenase E3 deficiency Alpha-ketoglutarate dehydrogenase complex Pyruvate dehydrogenase complex Pathogenic mutation Reactive oxygen species (ROS) X-ray crystallography Structure
E3 Deficiency—the Disease, Enzyme, and Affected Multienzyme Complex Functions
(Dihydro)lipoamide dehydrogenase (LADH, E3; gene: dld) deficiency is an often prematurely lethal rare autosomal recessive genetic disorder ; the highest carrier rate (1:94-1:110, G194C-hE3, h for human) has been found among Ashkenazi Jews with a disease frequency of 1:35,000–1:48,000 [2, 3]. The first and sole review on the molecular pathomechanisms of this disease was written by our laboratory . This disorder involves mainly neurological, cardiological, and hepatological manifestations whose symptoms generally arise very early in life. The phenotypic spectrum includes hyperammonemia, failure to thrive, hypotonia, encephalopathy, seizure, hepatomegaly, liver dysfunction, lactate acidosis, hypoglycemia, Leigh syndrome, developmental delay, hypertrophic cardiomyopathy, vision impairment/optic atrophy, ataxia, and microcephaly, among others [1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. Potentially lethal hepatological consequences, often together with encephalopathy and coagulopathy, may present in isolation and in adulthood [3, 17, 18, 19]. The severity of the clinical outcomes is due to the simultaneous defects of the mitochondrial E3-harboring multienzyme dehydrogenase complexes for alpha-ketoglutarate (KGDHc), pyruvate (PDHc), alpha-ketoadipate (KADHc), and branched-chain alpha-keto acids (BCKDHc); interestingly, the glycine cleavage system (GCS), which also contains the LADH protein, remains unaffected in E3-deficiency [1, 4, 20, 21, 22]. In the above dehydrogenase complexes the common E3 subunit catalyzes the re-oxidation of the dihydrolipoate (DHLA) moieties covalently linked to the respective E2 components and the reduction of NAD+ to NADH (LADH activity, forward reaction). Pathogenic gene variants include missense or nonsense mutations, splice site variants, and ones with (small) deletions/insertions; 14 disease-causing enzyme variants have been reported to date in the clinical literature [4, 6, 8].
Auxiliary Exacerbating Pathomechanisms
Clinical severity, more often than not, does not parallel the loss in LADH activity [4, 6, 8, 13, 20, 23, 24]. Recent results suggest that the missing clues could be (i) enhanced reactive oxygen species (ROS) production by various pathogenic hE3 mutants [20, 23, 24], particularly in acidosis , (ii) liberation of selected hE3 variants from the E3-tethering multienzyme complexes [25, 26, 27, 28, 29], and (iii) ROS generation by the E1–E2 subcomplex of the hKGDHc (when E3 is scarce) , principally in the course of acidosis .
ROS Generation by hE3, Pathogenic hE3 Mutants, and hE3-Harboring Multienzyme Complexes
Among all the mitochondrial alpha-keto (or 2-oxo) acid dehydrogenase complexes (OADHc), the KGDHc exhibits the most dominant ROS generation under pathologically relevant conditions [20, 21, 22, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42]. ROS generation by the KGDHc can occur in the forward catalytic direction in case the physiological electron acceptor NAD+ is scarce or absent (in vitro), or alternatively in the reverse reaction driven by a high NADH/NAD+ ratio [20, 30, 33, 34]; superoxide (as a primary ROS) is generated at the flavoenzyme E3 component [20, 33, 34] (see Scheme 1). The isolated E3 component [43, 44] is also capable of generating ROS, in an oxidase reaction, in either direction of the catalytic reaction [20, 24, 30, 45, 46, 47, 48, 49, 50, 51] (Scheme 1). The E3 subunit forms a functional (obligate), non-covalent homodimer that uses residues from both monomers for the physiological LADH activity [45, 52, 53, 54, 55], but not for the ROS-generating activity [56, 57, 58]; hE3 comprises four domains: a FAD-binding (1–149), a NAD+/NADH-binding (150–282), a central (283–350), and an interface domain (351–474). ROS generation in the reverse reaction of the isolated E3 component is stimulated in acidosis; this same pathological condition also enhances the ROS production in the reverse, but not in the forward reaction of KGDHc . Sensitivity to a decreasing pH of ROS-generation by isolated disease-causing hE3 mutants that display increased ROS-generating capacities in the reverse reaction was reported to be even more pronounced ; pathogenic substitutions which stimulated ROS production took place at the disulfide-exchange site (P453L), the dimerization surface (E340K, D444V), or the cofactor-binding site (G194C) . Calibrated gel filtration, molecular dynamics (MD) simulation, hydrogen–deuterium exchange mass spectrometry (HDX-MS), diffusion-ordered (DOSY) NMR, (soft-ionizing) nano-LC MS, and X-ray crystallography (see below) all confirmed that neither (mild) acidosis nor the hitherto investigated disease-causing homodimerization surface mutations led to monomerization of the E3 dimer [24, 25, 30, 59, 60, 61, 62]. Importantly, ROS production could also be stimulated by a relevant disease-causing hE3 mutant (G194C-hE3) when complexed to a multienzyme complex (hKGDHc) . The D444V-, G194C-, E340K-, R460G-, and R447G-hE3 pathogenic mutants were reported to oxidatively deteriorate the lipoic acid (LA) cofactors of the PDHc and KGDHc in a yeast model, and in case of D444V-hE3, in human homozygous fibroblasts . In mutants exhibiting stimulated ROS production, LADH activity was generally impaired in both catalytic directions. In P453L-hE3 the physiological activity was almost entirely lost while the ROS-generating activity became predominant, whereas in G194C-hE3 the LADH activity was not altered, but the ROS-producing capacity increased . For P453L-hE3 the clinical phenotype was very severe [16, 63] and the excessive ROS production was proposed to be a contributing factor to this . G194C-hE3 leads often to adult-onset manifestations, which is in accord with the retained LADH activity and the moderately enhanced ROS generation . FAD contents were almost entirely retained in D444V-hE3 and E340K-hE3, while G194C-hE3 and P453L-hE3 exhibited ~ 30% loss of FAD . Circular dichroism (CD) spectroscopy represented no significant overall structural alterations in the above four mutants . HDX-MS however detected significant changes in flexibility/exposure in the lipoic acid (LA) binding channel of P453L-hE3 and G194C-hE3, which could potentially result in stimulation of ROS production. HDX-MS data for D444V-hE3 and E340K-hE3 were rather inconclusive in terms of the mechanism of action of increased ROS production . MD simulation of 13 disease-causing hE3 mutants was also carried out [59, 60]; good correlation with HDX-MS results was reported for the P453L, K37E, G194C, I445M, and R460G substitutions .
Conclusions of the First Disease-Causing Mutant Structure (D444V-hE3) Relevant to Compromised Enzymatic Activity and ROS Generation
Crystal structures have been published for hE3 [28, 29, 52, 61, 64] and now also for the D444V-hE3 disease-causing mutant from our laboratory . High-resolution crystal structures have very recently been determined in our laboratory also for the P453L- (PDB ID: 6I4Z), G194C- (PDB ID: 6I4P), R460G- (PDB IDs: 6I4R and 6HG8), R447G- (PDB ID: 6I4S), I445M- (PDB ID: 6I4T), and G426E-hE3 (PDB ID: 6I4U) disease-causing variants and for hE3 at the hitherto highest 1.75 Å resolution (PDB ID: 6I4Q), however, the thorough and comparative analysis of these structures is still in progress. The already published and analyzed D444V-hE3 crystal structure demonstrated a shorter and wider H+/H2O-releasing channel when compared to the wild type structure. This channel is solvent accessible, leads to the active site and it is the continuation of the LA-binding substrate channel (Fig. 1); the H+/H2O channel appears to have catalytic roles in LADH function and perhaps ROS generation by hE3 . A drop in surface potential around the exit of this channel was also found when compared to the hE3 structure. Several helices and random coils form the above channel, the helices all pointing with the positive ends of their dipoles towards the active site; structural alterations in the channel-forming helices likely affect the active site via relayed helix dipole moment contributions. All the above mentioned structural alterations in D444V-hE3 hence indeed may lead to a drop in enzyme activity and the positive shift in ROS-generating capacity . Another indirect effect that might also have contribution to the pathological behaviors is an overall change in penetration through the channel upon structural changes, which was suggested altering the apparent redox potential of the FAD moiety [65, 66]; the redox status of FAD has direct influence on both the regular catalytic and ROS-generating activities. The C-terminus in D444V-hE3 was shown by HDX-MS to possess higher flexibility as compared to hE3 ; this effect could not be detected by crystallography, perhaps due to the cryogenic conditions. Since the C-terminus separates and hence forms connection between the LA-binding and the H+/H2O channels, any change in this region might also affect the LA-binding substrate channel, which is also implicated in both the normal catalytic action as well as superoxide generation [25, 61]. Since the disease-causing dimer interface substitutions (D444V, E340K, R447G, R460G, I445M) all take place in the vicinity of the H+/H2O channel (Fig. 2), considerably far from the LA-binding channel, the cofactor-binding sites, and the active site, this channel was connected to the potential existence of a generalized pathomechanism of human E3-deficiency for the disease-causing dimerization interface mutations .
Affinity Loss for Multienzyme Complexes and Respective Structural Conclusions for D444V-hE3
The affinity of E3 for the KGDHc proved to be low [67, 68, 69] and even lower in acidosis ; E3 binds ~ 30 times stronger to the PDHc [68, 70, 71]. Several experimental evidence suggest that E1, and not E2, would directly bind E3 in the KGDHc [72, 73, 74]. LADH can also exist as a liberated protein in vivo [30, 68, 75, 76, 77, 78]; it is the most abundant flavoprotein in brain and muscle mitochondria . Several disease-causing hE3 variants (R447G-, D444V-, R460G-, and E340K-hE3) exhibit significantly impaired affinity for the hPDHc leading to greatly compromised overall hPDHc activities [25, 26, 27, 29]. The D444V-hE3 crystal structure demonstrated a drop in surface potential over the entire protein molecule, while HDX-MS showed an enhanced flexibility on the surface where the E3-binding protein (E3BP) of hPDHc is tethered [25, 61]; both effects likely contribute to the compromised affinity for hPDHc. The D444V-hE3 crystal structure also confirmed previous experimental data on the lack of monomerization and FAD loss in this mutant .
ROS Generation by the E1–E2 Subcomplex of the hKGDHc
In case hE3 is untethered from the hKGDHc, as is likely the case for several pathogenic variants and in acidosis, the E1-E2 subcomplex is potentially also capable of generating ROS at a very considerable rate in the forward catalytic direction  (Scheme 1). Thus, under such conditions, ROS production might proceed simultaneously from E3 (principally in the reverse catalytic direction) as well as from the E1-E2 subcomplex (in the forward catalytic direction), provided that substrate provision is sufficient [4, 20]; an intact population of KGDHc may still retain some overall activity , unless the LA prosthetic group suffers oxidative damage . Since the E2 (dihydrolipoamide succinyltransferase) component could potentially also be targeted against ROS generation, we very recently determined its cryo-electronmicroscopic structure; we were able to detect residues 218–453 at 3.31 Å resolution (PDB ID: 6H05), which in a further and still progressing stage of structure determination appears to develop to be 2.9 Å resolution (unpublished result).
In conclusion, human E3-deficiency is still an incurable and hardly manageable disease, which might be taken under better control by dietary restrictions, nutritional support, correction of metabolic acidosis and coagulopathy, administration of flavins, lipoic acid, thiamine, trial of dichloroacetate, for selected and respective cases [1, 4]. Since in many cases with impaired affinity for multienzyme complexes the residual hE3 activity is much higher than the residual overall multienzyme complex activity, with the high-resolution crystal structures in hand, adaptor molecules could potentially be developed to retether the respective pathogenic hE3 mutants to their harboring multienzyme complexes; although this might only be a limited therapeutic solution, the approach can potentially be advantageous for selected patients. Development of specific ROS generation inhibitors against selected hE3 mutants and the E2 subunit of the hKGDHc, besides a general antioxidant therapy, might also be a valid approach towards possible therapeutic solutions. Besides gene therapy, which still requires substantial development to be efficient and completely safe, enzyme replacement therapy might gain potentials in the treatment of human E3 deficiency in the near future [80, 81].
Open access funding provided by Semmelweis University (SE). Financial support is acknowledged from the Hungarian Academy of Sciences (MTA Grant 02001 to Vera Adam-Vizi), the Hungarian Scientific Research Fund (OTKA Grant 112230 to V.A.-V.), and the Hungarian Brain Research Program (Grant 2017-1.2.1-NKP-2017-00002 to V.A.-V.). The author thanks Eszter Szabo for the figures and her valuable comments and Prof. Vera Adam-Vizi for her long time support.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
- 1.Quinonez SC, Thoene JG (2014) Dihydrolipoamide Dehydrogenase Deficiency. In: Pagon RA, Adam MP, Ardinger HH et al (ed) GeneReviews® [Internet] 1993–2016. University of Washington, Seattle, Seattle (WA) pp 1–37Google Scholar
- 5.Carrozzo R, Torraco A, Fiermonte G, Martinelli D, Di Nottia M, Rizza T, Vozza A, Verrigni D, Diodato D, Parisi G, Maiorana A, Rizzo C, Pierri CL, Zucano S, Piemonte F, Bertini E, Dionisi-Vici C (2014) Riboflavin responsive mitochondrial myopathy is a new phenotype of dihydrolipoamide dehydrogenase deficiency. The chaperon-like effect of vitamin B2. Mitochondrion 18:49–57CrossRefPubMedGoogle Scholar
- 9.Odievre MH, Chretien D, Munnich A, Robinson BH, Dumoulin R, Masmoudi S, Kadhom N, Rötig A, Rustin P, Bonnefont JP (2005) A novel mutation in the dihydrolipoamide dehydrogenase E3 subunit gene (DLD) resulting in an atypical form of alpha-ketoglutarate dehydrogenase deficiency. Hum Mutat 25:323–324CrossRefPubMedGoogle Scholar
- 10.Hong YS, Korman SH, Lee J, Ghoshal P, Qu Q, Barash V, Kang S, Oh S, Kwon M, Gutman A, Rachmel A, Patel MS (2003) Identification of a common mutation (Gly194Cys) in both Arab Moslem and Ashkenazi Jewish patients with dihydrolipoamide dehydrogenase (E3) deficiency: possible beneficial effect of vitamin therapy. J Inherit Metab Dis 26:816–818CrossRefPubMedGoogle Scholar
- 11.Grafakou O, Oexle K, van den Heuvel L, Smeets R, Trijbels F, Goebel HH, Bosshard N, Superti-Furga A, Steinmann B, Smeitink J (2003) Leigh syndrome due to compound heterozygosity of dihydrolipoamide dehydrogenase gene mutations. Description of the first E3 splice site mutation. Eur J Pediatr 162:714–718CrossRefPubMedGoogle Scholar
- 12.Cerna L, Wenchich L, Hansiková H, Kmoch S, Peskova K, Chrastina P, Brynda J, Zeman J (2001) Novel mutations in a boy with dihydrolipoamide dehydrogenase deficiency. Med Sci Monitor 7:1319–1325Google Scholar
- 17.Brassier A, Ottolenghi C, Boutron A, Bertrand AM, Valmary-Degano S, Cervoni JP, Chretien D, Arnoux JB, Hubert L, Rabier D, Lacaille F, de Keyzer Y, Di Martino V, de Lonlay P (2013) Dihydrolipoamide dehydrogenase deficiency: a still overlooked cause of recurrent acute liver failure and Reye-like syndrome. Mol Genet Metab 109:28–32CrossRefPubMedGoogle Scholar
- 20.Ambrus A, Nemeria NS, Torocsik B, Tretter L, Nilsson M, Jordan F, Adam-Vizi V (2015) Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components. Free Radic Biol Med 89:642–650CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Nemeria NS, Gerfen G, Yang LY, Zhang X, Jordan F (2018) Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine, l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro. Biochim Biophys Acta-Bioenerg 1859:932–939CrossRefPubMedGoogle Scholar
- 22.Nemeria NS, Gerfen G, Nareddy PR, Yang LY, Zhang X, Szostak M, Jordan F (2018) The mitochondrial 2-oxoadipate and 2-oxoglutarate dehydrogenase complexes share their E2 and E3 components for their function and both generate reactive oxygen species. Free Radic Biol Med 115:136–145CrossRefPubMedGoogle Scholar
- 25.Ambrus A, Wang JJ, Mizsei R, Zambo Z, Torocsik B, Jordan F, Adam-Vizi V (2016) Structural alterations induced by ten disease-causing mutations of human dihydrolipoamide dehydrogenase analyzed by hydrogen/deuterium-exchange mass spectrometry: implications for the structural basis of E3 deficiency. Biochim Biophys Acta-Mol Basis Dis 1862:2098–2109CrossRefGoogle Scholar
- 42.Fisher-Wellman KH, Gilliam LAA, Lin CT, Cathey BL, Lark DS, Neufer PD (2013) Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload. Free Radic Biol Med 65:1201–1208CrossRefPubMedPubMedCentralGoogle Scholar
- 59.Ambrus A, Mizsei R, Adam-Vizi V (2015) Structural alterations by five disease-causing mutations in the low-pH conformation of human dihydrolipoamide dehydrogenase (hLADH) analyzed by molecular dynamics—implications in functional loss and modulation of reactive oxygen species generation by pathogenic hLADH forms. Biochem Biophys Reports 2:50–56CrossRefGoogle Scholar
- 64.Brautigam CA, Wynn RM, Chuang JL, Naik MT, Young BB, Huang TH, Chuang DT (2011) Structural and thermodynamic basis for weak interactions between dihydrolipoamide dehydrogenase and subunit-binding domain of the branched-chain alpha-ketoacid dehydrogenase complex. J Biol Chem 286:23476–23488CrossRefPubMedPubMedCentralGoogle Scholar
- 72.Zhou J, Yang L, Ozohanics O, Zhang X, Wang J, Ambrus A, Arjunan P, Brukh R, Nemeria NS, Furey W, Jordan F (2018) A multipronged approach unravels unprecedented protein-protein interactions in the human 2-oxoglutarate dehydrogenase multienzyme complex. J Biol Chem 293:19213–19227CrossRefPubMedGoogle Scholar
- 74.McCartney RG, Rice JE, Sanderson SJ, Bunik V, Lindsay H, Lindsay JG (1998) Subunit interactions in the mammalian alpha-ketoglutarate dehydrogenase complex - Evidence for direct association of the alpha-ketoglutarate dehydrogenase and dihydrolipoamide dehydrogenase components. J Biol Chem 273:24158–24164CrossRefPubMedGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.