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

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...

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Autores principales: Szabo, Eszter, Nemes-Nikodem, Eva, Vass, Krisztina Rubina, Zambo, Zsofia, Zrupko, Eszter, Torocsik, Beata, Ozohanics, Oliver, Nagy, Balint, Ambrus, Attila
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341545/
https://www.ncbi.nlm.nih.gov/pubmed/37446004
http://dx.doi.org/10.3390/ijms241310826
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author Szabo, Eszter
Nemes-Nikodem, Eva
Vass, Krisztina Rubina
Zambo, Zsofia
Zrupko, Eszter
Torocsik, Beata
Ozohanics, Oliver
Nagy, Balint
Ambrus, Attila
author_facet Szabo, Eszter
Nemes-Nikodem, Eva
Vass, Krisztina Rubina
Zambo, Zsofia
Zrupko, Eszter
Torocsik, Beata
Ozohanics, Oliver
Nagy, Balint
Ambrus, Attila
author_sort Szabo, Eszter
collection PubMed
description 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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spelling pubmed-103415452023-07-14 Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase Szabo, Eszter Nemes-Nikodem, Eva Vass, Krisztina Rubina Zambo, Zsofia Zrupko, Eszter Torocsik, Beata Ozohanics, Oliver Nagy, Balint Ambrus, Attila Int J Mol Sci Article 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. MDPI 2023-06-28 /pmc/articles/PMC10341545/ /pubmed/37446004 http://dx.doi.org/10.3390/ijms241310826 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Szabo, Eszter
Nemes-Nikodem, Eva
Vass, Krisztina Rubina
Zambo, Zsofia
Zrupko, Eszter
Torocsik, Beata
Ozohanics, Oliver
Nagy, Balint
Ambrus, Attila
Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase
title Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase
title_full Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase
title_fullStr Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase
title_full_unstemmed Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase
title_short Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase
title_sort structural and biochemical investigation of selected pathogenic mutants of the human dihydrolipoamide dehydrogenase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341545/
https://www.ncbi.nlm.nih.gov/pubmed/37446004
http://dx.doi.org/10.3390/ijms241310826
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