Organisms across all kingdoms of life crucially rely on the high-energy metabolite acetyl-CoA in the cellular cytosol to fuel pivotal biochemical processes such as fatty acid and cholesterol synthesis, and protein acetylation. However, the metabolic availability of acetyl-CoA is by no means a sinecure. Acting as a metabolic hub, ATP citrate lyase (ACL) harnesses the hydrolysis of ATP and metabolically generated citrate and coenzyme A to catalyze a sequence of reactions that utilize highenergy adducts to yield acetyl-CoA. Indeed, the remarkably conserved domain organization of prokaryotic and eukaryotic ACLs and its manifestation into giant enzymatic machines of 0.5 MDa is testament to the intricacies of molecular evolution. Yet despite the omnipresence of ACL enzymes across all kingdoms of life, the elucidation of the structural and molecular basis of their function and regulation has constituted a daunting task for structural biologists and biochemists for decades. My research program is fuelled by groundbreaking work in my team towards the first structural snapshots of a bacterial ACL holoenzyme and a related citryl-CoA lyase, showing that the secrets of such metabolic hubs, including human ACL, have come within reach.In this ERC project, I propose an integrative research effort combining structural, biophysical and biochemical approaches to (1) unravel the multistep reaction mechanism for citrate cleavage and (2) elucidate the associated functional and regulatory plasticity of the ACL holoenzyme. (3) In addition, I will dissect regulatory ACL interaction networks by coupling interaction studies to cellular assays to generate a comprehensive view of the cellular mechanisms underlying the spatiotemporal control of ACL activity. The results of this ERC project will provide the necessary structural platform to facilitate therapeutic targeting of human ACL, responding to rapidly growing evidence about its relevance in large-scale metabolic diseases and cancer.