Molecular Sciences - Posters L
Krisztina Rubina Vass*, Zsófia Zámbó*, Éva Nemes-Nikodém, Eszter Szabó, Bálint Nagy, Attila Ambrus
Department of Biochemistry, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
*these authors contributed equally to this work
Background: The α-ketoglutarate dehydrogenase complex (KGDHc) is rate-limiting in the Krebs cycle. It consists of multiple copies of three subunits, E1-3; E3 is common to all the mitochondrial α-keto acid dehydrogenase complexes. The hKGDHc is considered to be a major producer and a sensitive target of reactive oxygen species (ROS). The enzymatic dysfunction of and ROS generation by the hKGDHc are heavily implicated in neurodegenerative diseases, ischemia-reperfusion, lactic acidosis, E3-deficiency, among others. ROS are produced by the intact hKGDHc at the FAD prosthetic group in E3. Pathogenic mutations of hE3 lead to the severe, often prematurely lethal, human disease E3 deficiency.
Aims: We wished to explore the structural bases of the altered enzymatic activities and, in selected cases, enhanced ROS formation of hE3 mutants. We also wished to investigate the effects of selected pathogenic hE3 mutations on the hKGDHc. The present results complement previous studies.
Methods: Subunits of the hKGDHc (E1-3) and pathogenic/active-site mutants of the hLADH were heterologously expressed in E. coli (pET52b+/BL21(DE3)) and purified. Site-directed mutagenesis was carried out using the QuikChange II kit. The constructs all included Strep affinity tags for purification in a single step. The structural and biochemical characterization involved X-ray crystallography, enzyme activity measurements (forward/reverse catalytic directions, ROS formation), calibrated gel filtration and FAD-content analysis.
Results and Conclusions: Among the 10 selected active-site mutants of hE3, the C50A and H452Q mutants exhibited drastic losses in the physiological LADH activity; interestingly, there could also be detected significant enhancements in the ROS-forming activity. In the cases of the G426E-, I318T-, I358T, I445M- and R460G-hE3 pathogenic mutants, we observed significant decreases in the FAD content. High-resolution crystal structures were determined for the I318T- and I358T-hE3 pathogenic variants. In relation to the structure of I318T-hE3, we also determined its specific activities: significant alterations relative to hE3 could only be detected in the reverse catalytic direction and ROS formation. The physiological activity of the recombinant hKGDHc got significantly compromised when assembled with the pathogenic P453L-hE3 mutant.
Funding: OTKA-K 143627, TKP2021-EGA-25