PhD Scientific Days 2024

Poster Session F - Molecular Medicine 3.

Recombinant Production and Characterization of Human Alpha-keto Acid Dehydrogenase Complex Subunits and Their Disease-relevant Variants

Text of the abstract

Introduction: The mitochondrial alpha-keto acid dehydrogenase enzyme complexes (KADHc) play pivotal roles in mitochondrial energy metabolism, catalyzing the oxidative decarboxylation of various alpha-keto acid substrates at the entry and within the Szent-Györgyi-Krebs cycle, as well as in the branched-chain amino acid metabolism, producing NADH and the corresponding acyl-CoA. Disease-relevant genetic mutations of these enzyme complexes generally lead to severe clinical manifestations, often involving damage to the nervous system, cardiovascular system, and/or liver, with premature death being a common outcome.
Aims: Our long-term objective is to investigate the structure and function of recombinant wild-type and disease-causing mutant hKADH complexes, as well as to uncover the mechanism of ROS formation by the complexes and their variants. To address this, first here we wished to express and purify the respective subunit protein products.
Methods: The hKADHc subunits and their pathogenic variants were heterologously expressed in E. coli and purified. Initially, the gene inserts underwent codon optimization for E. coli expression, followed by cloning into the pD454-SR plasmid and expression in BL21(DE3) E. coli cells. Subsequently, site-directed mutagenesis was carried out using the QuikChange II kit. The expression constructs contained N-terminal Twin-Strep or His affinity tags to enable efficient purification, generally in a single step. The protein products were characterized for sequence, integrity and purity by mass spectrometry and SDS-PAGE. To assess subunit-subunit interactions in the complexes, we also established a surface plasmon resonance protocol.
Results and Conclusions: Purification of R6C-, R43C- and R349C-hE1p, as well as G307E- and D237N-hE2k was successfully implemented. Production of the hE2p and hE1a subunits/components demands further development. We also already possess preliminary results on the subunit-subunit interactions in the hKGDHc.
Funding: OTKA-K 143627, TKP2021-EGA-25