Refolding of the human dihydrolipoamide dehydrogenase

Ambrus, Attila; Törőcsik, Beáta; Ádám-Vizi, Vera

doi:10.1016/j.bej.2009.03.004

Cited by 10 publications

(5 citation statements)

References 40 publications

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“…In the physiological (forward) direction LADH oxidizes the complex-bound dihydrolipoic acid moiety while generating NADH from NAD + . When the reverse reaction is modeled in vitro, isolated LADH [1,2] oxidizes NADH while reducing model substrates, like lipoic acid or lipoamide [3]. In both directions in the absence of added electron acceptors, O 2 is reduced by LADH to superoxide ("oxidase reaction" of LADH), the latter being a reactive oxygen species (ROS), which is then partly dismutated to H 2 O 2 and O 2 [3][4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Ambrus

Ádám-Vizi

2013

Archives of Biochemistry and Biophysics

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Human dihydrolipoamide dehydrogenase (LADH, E3) is a component in the pyruvate-, alpha-ketoglutarate-and branched-chain ketoacid dehydrogenase complexes and in the glycine cleavage system. The pathogenic mutations of LADH cause severe metabolic disturbances, called E3 deficiency that often involve cardiological and neurological symptoms and premature death. Our laboratory has recently shown that some of the known pathogenic mutations augment the reactive oxygen species (ROS) generation capacity of LADH, which may contribute to the clinical presentations. A recent report concluded that elevated oxidative stress generated by the above mutants turns the lipoic acid cofactor on the E2 subunits dysfunctional. In the present contribution we generated by molecular dynamics (MD) simulation the conformation of LADH that is proposed to be compatible with ROS generation. We propose here for the first time the structural changes, which are likely to turn the physiological LADH conformation to its ROS-generating conformation. We also created nine of the pathogenic mutants of the ROS-generating conformation and again used MD simulation to detect structural changes that the mutations induced in this LADH conformation. We propose the structural changes that may lead to the modulation in ROS generation of LADH by the pathogenic mutations.

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Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Ambrus

Ádám-Vizi

2013

Archives of Biochemistry and Biophysics

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“…It is of potential interest that the residual values for the FAD content and the catalytic activities correlated quite well for the G101del mutation. Our laboratory [ 26 ] and others [ 27 ] reported the importance of FAD in the folding and stabilization of LADH; hence, FAD loss might as well, at least in part, account for the compromised protein stability and/or folding in relevant cases.…”

Section: Discussionmentioning

confidence: 99%

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase

Szabo,

Nemes-Nikodem,

Vass

et al. 2023

IJMS

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Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the forward and reverse catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised. None of the above three variants lent themselves to an in-depth structural analysis via X-ray crystallography due to inherent protein instability. Crystal structures at 2.89 and 2.44 Å resolutions were determined for the I318T- and I358T-hLADH variants, respectively; structure analysis revealed minor conformational perturbations, which correlated well with the residual LADH activities, in both cases. For the dimer interface variants G426E-, I445M-, and R447G-hLADH, enzyme activities and FAD loss were determined and compared against the previously published structural data.

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“…70 µL 20 mM FAD and 33 µL 50 mg/mL avidin (IBA, Gottingen, Germany) were added to the cleared lysate. For affinity purification, a 5 mL Strep-Tactin Macroprep FPLC column (IBA, Gottingen, Germany) was applied on an AKTA Purifier 10 UPC FPLC system (GE Healthcare Biosciences AB, Uppsala, Sweden) according to our published protocol for hE3 purification [54, 67], which requires no further purification steps. Protein eluates were concentrated to 10–15 mg/mL using Amicon Ultracel centrifugation filtering tubes (MWCO=30 kDa; Millipore, Cork, Ireland) at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

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

Ambrus¹,

Wang²,

Mizsei³

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Pathogenic amino acid substitutions of the common E3 component (hE3) of the human alpha-ketoglutarate dehydrogenase and the pyruvate dehydrogenase complexes lead to severe metabolic diseases (E3 deficiency), which usually manifest themselves in cardiological and/or neurological symptoms and often cause premature death. To date, 14 disease-causing amino acid substitutions of the hE3 component have been reported in the clinical literature. None of the pathogenic protein variants has lent itself to high-resolution structure elucidation by X-ray or NMR. Hence, the structural alterations of the hE3 protein caused by the disease-causing mutations and leading to dysfunction, including the enhanced generation of reactive oxygen species by selected disease-causing variants, could only be speculated. Here we report results of an examination of the effects on the protein structure of ten pathogenic mutations of hE3 using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), a new and state-of-the-art approach of solution structure elucidation. On the basis of the results, putative structural and mechanistic conclusions were drawn regarding the molecular pathogenesis of each disease-causing hE3 mutation addressed in this study.

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Refolding of the human dihydrolipoamide dehydrogenase

Cited by 10 publications

References 40 publications

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase

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

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