PhD Scientific Days 2026

Molecular Medicine 2.

Heterologous Expression, Purification and Characterization of Mitochondrial Alpha-keto Acid Dehydrogenase Complex Subunits and Their Disease-relevant Mutant Variants

Name of the presenter

Zubánics-Nagy, Áron

Institute/workplace of the presenter

Institute of Biochemistry and Molecular Biology, Department of Biochemistry, Semmelweis University

Authors

Áron Zubánics-Nagy¹, Momen Sameh Elsayed Fadel ELKHESHEN¹, Wafa Mutasim Mohamedosman Alamin¹, Bálint Csuka-Nagy¹, Attila Ambrus¹
1: Institute of Biochemistry and Molecular Biology, Department of Biochemistry, Semmelweis University

Text of the abstract

Introduction: Mitochondrial α-keto acid dehydrogenase complexes play key roles in cellular metabolism and also display non-canonical (“moonlighting”) functions, like reactive oxygen species (ROS) generation. This enzyme family comprises the α-ketoglutarate (k), pyruvate (p), α-ketoadipate (a) and branched-chain α-keto acid (b) dehydrogenase complexes. They consist of three major subunits: the α-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide transacylase (E2), both specific to the complex, and (the shared) dihydrolipoamide dehydrogenase (E3). Mutations that hit the human (h) subunit enzymes often lead to clinically severe disorders and premature death.

Aims: The long-term objective of our study is to investigate the molecular pathology of relevant disease-causing variants via detailed structure-function analysis that involves state-of-the-art structural biology methods like X-ray crystallography, cryo-EM and NMR spectroscopy.

Methods: Heterologous expression of selected subunits and their pathogenic mutants was carried out in the E. coli BL21(DE3) host using either the pET-52b(+) or pD454-SR expression plasmid under optimized conditions. Gene inserts were synthesized after codon optimization and modified by site-directed mutagenesis; all of the inserts also coded for an N-terminal Twin-Strep-tag. Recombinant protein products were purified to homogeneity by affinity chromatography in a single step and analyzed by LDS–PAGE, the Bradford assay and CD spectroscopy (some mutants).

Results: In this study, we successfully generated the L64F- and Y102C-hE1p, D257A- and N126Y-hE1b, R411Q- and G685R-hE1a, and G214C-hE3 variants in their soluble homogeneous states (>90%); mutations were confirmed via DNA sequencing and mass spectrometry (for proteins). Assembling and measuring the overall activities of the wild-type multienzyme complexes were successful. A subunit-specific activity assay for the E1 proteins was also successfully optimized.

Conclusions: Preliminary results indeed show compromised catalytic activities for the investigated E1 mutants, which explain pathogenicity. Structure-function analysis of the respective disease-causing variants may facilitate the development of pharmacological intervention strategies via small-molecule drug design.

Funding: OTKA grant 143627 and TKP2021-EGA-25 grant (both to A.A.).