Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8,9,10,11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.
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Protein expression constructs generated in this study are available via the BCCM/GeneCorner Plasmid Collection (http://bccm.belspo.be) through the following accession codes: LMBP 11277 (pTrcHis2-hACLY), LMBP 11131 (pET-DUET-hACLY-A/B), LMBP 11132 (pET11a-Mco-ACLY-A/B), LMBP 11133 (pET11a-Hth-CCL), LMBP 11134 (pET-Duet-Hth-CCSα/β), LMBP 11125 (pET11a-Cli-ACLY-A/B), LMBP 11128 (pET15b-hCCL) and LMBP 11129 (pET15b-Cli-CCL). X-ray crystallographic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession codes 6HXH (hACLY-A/B in space group P1), 6QFB (hACLY-A/B in space group C2), 6HXI (M. concilii ACLY-A/B), 6HXJ (C. limicola ACLY-A/B), 6HXK (CCL module of hACLY, space group P212121), 6HXL (CCL module of hACLY, space group P21), 6HXM (CCL module of hACLY, space group C2221), 6HXN (CCL module of C. limicola ACLY, space group P3121), 6HXO (CCL module of C. limicola ACLY, space group P21), 6QCL (CCL module of C. limicola ACLY in complex with acetyl-CoA and l-malate), 6HXP (H. thermophilus CCL) and 6HXQ (H. thermophilus CCS). SAXS data and models have been deposited in the Small Angle Scattering Biological Data Bank with accession codes SASDE36, SASDE46 and SASDE56 for hACLY-A/B; SASDFA3, SASDFB3 and SASDFC3 for hACLY; and SASDE66, SASDE76 and SASDE86 for C. limocola ACLY-A/B. Source Data for the SEC–MALLS analysis of hACLY-A/B (Extended Data Fig. 1d) and for the enzymatic assays for hACLY and hACLY-A/B (Extended Data Fig. 1e) are available online. Data are available from the corresponding author(s) upon reasonable request.
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K.V. and Y.B. are post-doctoral research fellows of the Research Foundation – Flanders (FWO) (fellowships 12A5517N and 12S0519N). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017). This work was supported by grants from the FWO to K.V. (1524918N), a Concerted Research Action (GOA) grant from Ghent University to S.N.S. (BOF17-GOA-028), a Hercules Foundation infrastructure grant to S.N.S. (AUGE-11-029), a programme grant from the VIB to S.N.S., and the Horizon 2020 grants: Chap4Resp (to I.G., grant no. 647784), iNext (grant no. 653706) and CALIPSOplus (grant no. 730872). We acknowledge access to experimental facilities and technical support at the following synchrotron radiation facilities: PETRA III (beamlines P12, P13 and P14), SOLEIL (Proxima-2, Swing), ESRF (ID23-1, ID23-2 and ID30-B), SLS (PXI and PXIII). This work used the platforms of the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The electron microscope facility is supported by the Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), Fonds FEDER, the Centre National de la Recherche Scientifique (CNRS), the CEA, the University of Grenoble, EMBL, and the GIS-Infrastrutures en Biologie Sante et Agronomie (IBISA).
Nature thanks Frank M. Raushel and the other anonymous reviewer(s) for their contribution to the peer review of this work.