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

Nature Structural & Molecular Biology volume 28, pages 636–638 (2021)Cite this article

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Matters Arising to this article was published on 22 July 2021

The Original Article was published on 23 December 2019

arising from: X. Wei, K. Schultz, G. A. Bazilevsky, A. Vogt & R. Marmorstein. Nature Structural & Molecular Biology https://doi.org/10.1038/s41594-019-0351-6 (2020).

Recent structural studies on ACLY1,2,3,4, a central enzyme for the biosynthesis of the critical metabolite acetyl-coenzyme A (CoA)5,6,7,8,9,10,11, provided a long-awaited framework to propose the catalytic mechanism of the enzyme1,2,3. ACLY holoenzymes across all kingdoms of life adopt conserved tetrameric structures of roughly 500 kDa comprising subunits featuring an N-terminal acyl-CoA synthetase homology (ASH) module and a C-terminal citrate synthase homology (CSH) module. The catalytic itinerary of ACLY starts in the ASH module where ATP-driven autophosphorylation leads to activation of citrate to citryl-phosphate. This enables the reaction with CoA to yield the high-energy intermediate citryl-CoA. Shuttling of citryl-CoA to the CSH module of ACLY, facilitated by flipping of the long pantothenyl-arm of citryl-CoA to position the citryl-thioester moiety in the CSH active site, sets the stage for the last step of the reaction, namely the retro-aldol cleavage of citryl-CoA into oxaloacetate and acetyl-CoA (Extended Data Fig. 1a,b). A study by Wei et al.12,13 claimed that both the formation and the retro-aldol cleavage of the citryl-CoA intermediate are catalyzed in the ASH module of ACLY instead, with the CSH module not serving a catalytic role. In light of previous publications1,2,3,4 on ACLY and new data presented here, we argue that this claim is incorrect and that the study by Wei et al.12,13 suffers from critical issues, discussed below.

  1. 1.

    The tetrameric CSH module in ACLY is evolutionarily related to standalone citryl-CoA lyase, which catalyzes the conversion of citryl-CoA into oxaloacetate and acetyl-CoA1,14. The CSH module is also homologous to citrate synthase, and ligand-bound crystal structures at high resolution show that the CSH module of ACLY and citrate synthase adopt equivalent closed active site configurations poised for the retro-aldol cleavage of (3S)-citryl-CoA1 (Extended Data Fig. 1c). The catalytic importance of active site residues in the CSH module in ACLY was shown by mutagenesis3. Wei et al. also show that the active site residues of the CSH module—which are conserved in citrate synthase—are critical for enzymatic activity, as these ACLY mutants are catalytically dead (Extended Data Fig. 1c,d). These enzymological data cannot be reconciled by the main conclusion by Wei et al. that all chemistry of the ACLY reaction mechanism occurs in the ASH module.

  2. 2.

    Based on structural analysis by single-particle cryogenic-electron microscopy of human ACLY incubated with the reaction products oxaloacetate and acetyl-CoA (PDB 6UV5 and 6UI9) and the ensuing modeling of oxaloacetate and the acetyl-thioester moiety of acetyl-CoA in the ASH module, Wei et al. claim to have demonstrated that the ASH module is the site for both the synthesis and cleavage of citryl-CoA, and that the CSH module is catalytically inactive. At the outset, the observation of exogenously added reaction products bound in an enzyme active site does not demonstrate that the reaction products can be enzymatically generated in that particular active site. Wei et al. observed exogenously added oxaloacetate in the CSH module of ACLY as well. Thus, based on such data, one cannot conclude that oxaloacetate is enzymatically generated in either of the two active sites. The same applies for the acetyl-thioester moiety of bound acetyl-CoA, which as modeled by Wei et al. can orient toward the ASH and CSH active sites alike (PDB 6UV5, Extended Data Fig. 2a). In addition, the bound ligands presented by Wei et al. have been modeled in weak and often discontinuous density at low resolution, and do not engage in bona fide interactions with protein atoms in the active site. This is particularly exemplified in PDB 6UV5 and 6UI9, where the experimental density for the acetyl-thioester moieties of acetyl-CoA in the ASH active site is very poorly defined. Notably, certain modeled acetyl-CoA and CoA ligands display fundamental stereochemical errors (Supplementary Fig. 1). Thus, based on these structures, no conclusions can be made about the catalytic competence of the ACLY active sites (Extended Data Fig. 2a,b).

  3. 3.

    The structure for ACLY in complex with CoA reported by Wei et al. (PDB 6POE) shows how the CoA-binding domain of the CSH module can rotate to shuttle the citryl-CoA reaction intermediate and close the CSH active site to enable production of acetyl-CoA (Extended Data Fig. 1e), in full agreement with previous proposals for the ACLY reaction mechanism1,2,3. Yet, Wei et al. did not relate these conformational changes to the formation of a closed CSH active site or consider the catalytic role of the CSH module in the retro-aldol cleavage of citryl-CoA.

  4. 4.

    Wei et al. propose to have trapped a phospho-citryl-CoA reaction intermediate bound to an ACLY-E599Q mutant (ligand QHD in PDB 6UUW). This high-energy, short-lived reaction intermediate cannot be reliably identified in the experimental density (Extended Data Fig. 2c). Moreover, the stereochemistry of the modeled ligand QHD is incompatible with that of the ACLY reaction mechanism, which proceeds via (3S)-citryl-CoA4,8,10. Therefore, ligand QHD is incorrect as was the originally presented model for phospho-citryl-CoA by Wei et al.12,13 (Extended Data Fig. 2d).

  5. 5.

    Based on the structure of the ACLY-E599Q mutant in complex with ligand QHD, Wei et al. put forth a catalytic role for E599 in the cleavage of citryl-CoA. Such a proposal is enzymologically unfounded because phospho-citryl-CoA is a tetrahedral reaction intermediate in the third step of the acyl-CoA synthetase reaction toward citryl-CoA (Extended Data Fig. 1a). Thus, even if the presence of bound phospho-citryl-CoA in the ASH module could be shown robustly, it would merely suggest that residue E599 plays a role in the acyl-CoA synthetase reaction toward citryl-CoA, as previously proposed4. Furthermore, the enzymology data presented by Wei et al. cannot provide the information required to support the role of E599 in the citryl-CoA cleavage reaction because the experimental set-up chosen by the authors follows the overall reaction, rather than formation of an intermediate.

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Fig. 1: The CSH module of ACLY is catalytically active.
Fig. 2: Structural basis for shuttling of enzymatically generated (3S)-citryl-CoA from the ASH module to the CSH module of ACLY.

Data availability

X-ray crystallographic coordinates and structure factors have been deposited in wwPDB with accession codes PDB 6Z2H (CSH module of human ACLY) and PDB 6ZNW (archaeal ACLY). Raw data for the enzymatic assays are available via the online version of this paper. Other datasets used in this study are publicly available in the wwPDB (entries PDB 6HXH, 6HXI, 6HXL, 5CTS, 6UV5, 6UI9, 6POE and 6UUW) and the Electron Microscopy Data Bank (entries EMD-20902, EMD-20904, EMD-20783 and EMD-20413).

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Acknowledgements

We thank the staff of beamlines P14 (Petra III, Deutsches Elektronen-Synchrotron) and Proxima 2A (SOLEIL) for their technical support and beamtime allocation. This project was supported by a grant from Research Foundation Flanders to K.V. (no. G0G0619) and a Ghent University grant to S.N.S. (no. BOF17-GOA-028). S.N.S. is a principal investigator of the VIB (Belgium).

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Authors and Affiliations

  1. VIB-UGent Center for Inflammation Research, Ghent, Belgium

    Kenneth Verstraete, Koen H. G. Verschueren, Ann Dansercoer & Savvas N. Savvides

  2. Unit for Structural Biology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Kenneth Verstraete, Koen H. G. Verschueren, Ann Dansercoer & Savvas N. Savvides

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  1. Kenneth Verstraete
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Contributions

A.D. and K.V. purified proteins. K.V. and K.H.G.V. carried out structural studies and analysis with input from S.N.S. K.V. and S.N.S. wrote the paper.

Corresponding authors

Correspondence to Kenneth Verstraete or Savvas N. Savvides.

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Competing interests

K.V. and S.N.S. are listed as coinventors on a patent filed with the European Patent Office entitled ‘Crystal structure of ATP Citrate Lyase and uses thereof’ (EP19161520.2). K.H.G.V. and A.D. declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Structural basis for the retro-aldol cleavage of citryl-CoA by the CSH-module of ACLY.

a, The ACLY-catalyzed reaction proceeds via four steps. The first three steps lead up to the formation of citryl-CoA from ATP, citrate and CoA. In the fourth step, citryl-CoA undergoes retro-aldol cleavage to generate the reaction products oxaloacetate and acetyl-CoA. b, An overlay of CoA- and citrate-bound crystal structures for full-length human ACLY and its isolated CSH-module shows how the citryl-CoA intermediate can be shuttled from the ASH-active site towards the CSH-active site for retro-aldol cleavage. The CoA-binding domain of the CSH-module can rotate towards an open (white) or closed (pink) CSH-active site configuration. c, Closed active site configuration of the CSH-module of human ACLY in complex with CoA and citrate (PDB 6hxl) overlaid with the closed active site configuration of chicken citrate synthase (CS) in complex with carboxymethyl-CoA and oxaloacetate (grey carbon atoms, PDB 5cts). Residue numbers between brackets refer to the residue numbers of citrate synthase. The figures in panels b and c are adapted from Verschueren et al., (ref. 1). d, Enzymatic activity for human ACLY active site mutants reproduced from Fig. 1f in Wei et al.13 e, Overlay between chains A + C and chains B + D in PDB 6poe based on the superposition of the core of the CSH-module (residues 837–935 and residues 1055–1101; r.m.s.d.-value = 0.198 Å for 256 aligned C-alpha atoms). The superposition shows that in ACLY, bound CoA can adopt an extended conformation (white) pointing to the ASH active site, or a collapsed conformation (green) pointing to the CSH-active site. In the collapsed conformation, the CoA-binding domain (magenta) is rotated to form a closed CSH-active site configuration analogous to citrate synthase.

Extended Data Fig. 2 Ligands as modelled by Wei et al. and their corresponding experimental density.

a, Alternative conformations of acetyl-CoA (chain A, residue 1202) - as modelled by Wei et al. in PDB 6uv5 - point to ASH and CSH active sites, and oxaloacetate is modelled in both the ASH and CSH active sites. The author-recommend contour level in the EMD-databank is 0.012 (entry EMD-20904). b, Acetyl-CoA and oxaloacetate as modelled by Wei et al.13 in PDB 6ui9. Acetyl-CoA is pointing to the ASH-active site, and oxaloacetate is modelled both in the ASH- and CSH-active sites. The author-recommend contour level in the EMD-databank is 0.010 (entry EMD-20783). c, Experimental density for ligand QHD in PDB 6uuw. The black arrow indicates the poor connectivity between the phospho-citryl and CoA moieties. In panel a-c, the experimental ligand density is shown at different contour levels and carved around the ligand with a radius of 2 Å. d, The stereochemistry of the chiral carbon atom of the citryl-moiety in phosphocitryl-CoA as proposed by Wei et al.13 is incompatible with the ACLY-catalyzed reaction.

Supplementary information

Supplementary Information

Supplementary Note 1, Figs. 1–3 and Table 1.

Reporting Summary

Source data

Source Data Fig. 1

Raw data for the enzymatic assay.

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Verstraete, K., Verschueren, K.H.G., Dansercoer, A. et al. Acetyl-CoA is produced by the citrate synthase homology module of ATP-citrate lyase. Nat Struct Mol Biol 28, 636–638 (2021). https://doi.org/10.1038/s41594-021-00624-3

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