Mechanism, regulation and structure of human pyruvate dehydrogenase complex
by Rahul Shaha
Date of Examination:2025-04-25
Date of issue:2025-06-23
Advisor:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Kai Tittmann
Referee:Dr. Sonja Lorenz
Persistent Address:
http://dx.doi.org/10.53846/goediss-11323
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Abstract
English
The human pyruvate dehydrogenase complex (hPDHc) catalyzes the irreversible conversion of pyruvate to acetyl-CoA, linking glycolysis to the citric acid cycle - a central hub of cellular metabolism. This reaction proceeds through three sequential steps catalyzed by the E1, E2, and E3 subunits, with E1 (a thiamine diphosphate-dependent enzyme) initiating the process through pyruvate decarboxylation and reductive acetylation. Despite extensive studies, the detailed reaction mechanism and regulatory control of E1 remain incompletely understood, partly due to a lack of structural evidence for key reaction intermediates. Additionally, the mechanism by which site 2 and site 3 phosphorylation inactivates E1 is not fully established. In this thesis, we present the first crystal structures of human E1 (hE1) bound to pyruvate and a modified lipoyllysine, capturing key reaction intermediates in the wildtype enzyme. Our structural data reveal that the two active sites of hE1 exhibit asymmetry throughout catalysis, evident in both reaction intermediate distribution and loop conformations between the active sites. Furthermore, we propose that pyruvate decarboxylation is triggered in the presence of the lipoyl domain, as supported by intermediate distribution analysis and hE1 structures capturing a covalent adduct of lipoyllysine and ThDP. Combining structural and enzyme kinetics data, we suggest that H263 acts as the proton donor during reductive acetylation. MD simulations further strengthen the role of H263 as proton donor in hE1 Lipoyl domain complex. Structural and kinetic studies of site 2 and site 3 phosphomimic mutants show that site 2 phosphorylation disrupts catalytic competence, while site 3 phosphorylation reduces ThDP binding affinity, providing insights into PDHc regulation. In the second part of this study, we present previously unrecognized regulatory mechanisms of hPDHc by the metabolites dephospho-coenzyme A and octanoyl-coenzyme A, supported by X-ray structures of the E2 and E3 subunits in complex with these molecules. For the first time, we provide structural evidence that dephospho-coenzyme A directly targets the NAD+ site of E3, while octanoyl-CoA binds within the coenzyme A channel of E2, revealing a novel mode of PDHc regulation. Activation of hPDHc is crucial for treating metabolic disorders. In this study, we characterized the mechanism of activation of hPDHc by the synthetic molecule activators discovered in collaboration with Rutter’s lab. Our activity assays demonstrate that these effector molecules enhance hPDHc activity in both in vitro and in vivo conditions. Kinetic analyses indicate that these molecules facilitate E1-E2 binding and relieve feedback inhibition imposed by acetylCoA and octanoyl-CoA, thereby enhancing hPDHc activity. Cryo-EM structures of the E2 core 2 in complex with effector molecules reveal that these compounds bind at the lipoyl entry site, possibly influencing subunit dynamics and enzyme assembly. These findings provide key mechanistic insights into hPDHc reaction mechanism and regulation, revealing potential therapeutic targets for metabolic disorders involving hPDHc dysfunction, such as hPDHc deficiency and lactic acidosis. By identifying small-molecule activators, this work lays the foundation for novel strategies to modulate hPDHc activity and restore metabolic balance in disease states.
Keywords: human pyruvate dehydrogenase complex (hPDHc); Thiamine diphosphate; Pyruvate dehydrogenase E1; Reductive acetylation; Phosphorylation; Pyruvate dehydrogenase complex activators
