Acetyl-CoA is produced by the citrate synthase homology module of ATP-citrate lyase

Recent structural studies on ACLY1–4, a central enzyme for the biosynthesisof the critical metabolite acetyl-coenzyme A (CoA)5–11,provided a long-awaited framework to propose the catalytic mechanismof the enzyme1–3. ACLY holoenzymes across all kingdomsof life adopt conserved tetrameric structures of roughly 500 kDacomprising subunits featuring an N-terminal acyl-CoA synthetasehomology (ASH) module and a C-terminal citrate synthase homology(CSH) module. The catalytic itinerary of ACLY starts in theASH module where ATP-driven autophosphorylation leads to activationof citrate to citryl-phosphate. This enables the reaction withCoA to yield the high-energy intermediate citryl-CoA. Shuttling ofcitryl-CoA to the CSH module of ACLY, facilitated by flipping of thelong pantothenyl-arm of citryl-CoA to position the citryl-thioestermoiety in the CSH active site, sets the stage for the last step of thereaction, namely the retro-aldol cleavage of citryl-CoA into oxaloacetateand acetyl-CoA (Extended Data Fig. 1a,b). A study by Weiet al.12,13 claimed that both the formation and the retro-aldol cleavageof the citryl-CoA intermediate are catalyzed in the ASH moduleof ACLY instead, with the CSH module not serving a catalytic role.In light of previous publications1–4 on ACLY and new data presentedhere, we argue that this claim is incorrect and that the study byWei et al.12,13 suffers from critical issues, discussed below