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The structural basis of fatty acid elongation by the ELOVL elongases

Nature Structural & Molecular Biology volume 28, pages 512–520 (2021)Cite this article

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Abstract

Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson’s, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy.

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Fig. 1: Overall structure of ELOVL7.
Fig. 2: Heterologously expressed ELOVL7 is covalently bound to a 3-keto acyl-CoA.
Fig. 3: Acyl chain– and CoA-binding sites.
Fig. 4: Catalytic site around the HxxHH motif.
Fig. 5: Proposed ping-pong mechanism and evidence for a covalent acyl-enzyme intermediate.
Fig. 6: Covalent acyl-enzyme intermediate is formed on substrate reaction at H150.
Fig. 7: Model for malonyl-CoA binding at the decarboxylation site.

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Data availability

Atomic coordinates and structure factors for the reported crystal structure are deposited in the Protein Data Bank under accession code PDB 6Y7F.

References

  1. Leonard, A. E., Pereira, S. L., Sprecher, H. & Huang, Y.-S. Elongation of long-chain fatty acids. Prog. Lipid Res. 43, 36–54 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Sassa, T. & Kihara, A. Metabolism of very long-chain fatty acids: genes and pathophysiology. Biomol. Ther. (Seoul) 22, 83–92 (2014).

    Article  CAS  Google Scholar 

  3. Ohno, Y. et al. ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis. Proc. Natl Acad. Sci. USA 107, 18439–18444 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Imgrund, S. et al. Adult ceramide synthase 2 (CERS2)-deficient mice exhibit myelin sheath defects, cerebellar degeneration, and hepatocarcinomas. J. Biol. Chem. 284, 33549–33560 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Isokawa, M., Sassa, T., Hattori, S., Miyakawa, T. & Kihara, A. Reduced chain length in myelin sphingolipids and poorer motor coordination in mice deficient in the fatty acid elongase Elovl1. FASEB Bioadv 1, 747–759 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sassa, T. et al. Impaired epidermal permeability barrier in mice lacking Elovl1, the gene responsible for very-long-chain fatty acid production. Mol. Cell. Biol. 33, 2787–2796 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Li, W. et al. Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice. Int J. Biol. Sci. 3, 120–128 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Harkewicz, R. et al. Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function. J. Biol. Chem. 287, 11469–11480 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Pewzner-Jung, Y. et al. A critical role for ceramide synthase 2 in liver homeostasis: II. insights into molecular changes leading to hepatopathy. J. Biol. Chem. 285, 10911–10923 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Jakobsson, A., Westerberg, R. & Jacobsson, A. Fatty acid elongases in mammals: their regulation and roles in metabolism. Prog. Lipid Res. 45, 237–249 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Toke, D. A. & Martin, C. E. Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J. Biol. Chem. 271, 18413–18422 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Oh, C.-S., Toke, D. A., Mandala, S. & Martin, C. E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation. J. Biol. Chem. 272, 17376–17384 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Lee, S. H., Stephens, J. L. & Englund, P. T. A fatty-acid synthesis mechanism specialized for parasitism. Nat. Rev. Microbiol. 5, 287–297 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Ramakrishnan, S. et al. Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287, 4957–4971 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Haslam, T. M. & Kunst, L. Extending the story of very-long-chain fatty acid elongation. Plant Sci. 210, 93–107 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Venegas-Calerón, M., Beaudoin, F., Sayanova, O. & Napier, J. A. Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase. J. Biol. Chem. 282, 2996–3003 (2007).

    Article  PubMed  Google Scholar 

  17. Zhang, K. et al. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat. Genet. 27, 89–93 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Cadieux-Dion, M. et al. Expanding the clinical phenotype associated with ELOVL4 mutation: study of a large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia. JAMA Neurol. 71, 470–475 (2014).

    Article  PubMed  Google Scholar 

  19. Di Gregorio, E. et al. ELOVL5 mutations cause spinocerebellar ataxia 38. Am. J. Hum. Genet. 95, 209–217 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Moon, Y. A., Hammer, R. E. & Horton, J. D. Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice. J. Lipid Res. 50, 412–423 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Matsuzaka, T. et al. Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance. Nat. Med. 13, 1193–1202 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Tamura, K. et al. Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism. Cancer Res. 69, 8133–8140 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Tolkach, Y. et al. Signatures of adverse pathological features, androgen insensitivity and metastatic potential in prostate cancer. Anticancer Res. 35, 5443–5451 (2015).

    CAS  PubMed  Google Scholar 

  24. Zhang, X. & Wang, Y. Identification of hub genes and key pathways associated with the progression of gynecological cancer. Oncol. Lett. 18, 6516–6524 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Li, G. et al. Association of GALC, ZNF184, IL1R2 and ELOVL7 with Parkinson’s disease in southern Chinese. Front. Aging Neurosci. 10, 402 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Parisi, L. R. et al. Membrane disruption by very long chain fatty acids during necroptosis. ACS Chem. Biol. 14, 2286–2294 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Naganuma, T., Sato, Y., Sassa, T., Ohno, Y. & Kihara, A. Biochemical characterization of the very long-chain fatty acid elongase ELOVL7. FEBS Lett. 585, 3337–3341 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Denic, V. & Weissman, J. S. A molecular caliper mechanism for determining very long-chain fatty acid length. Cell 130, 663–677 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Hernandez-Buquer, S. & Blacklock, B. J. Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme. FEBS Lett. 587, 3837–3842 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Wang, H. et al. Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate. Nat. Struct. Mol. Biol. 22, 581–585 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. Röttig, A. & Steinbüchel, A. Acyltransferases in bacteria. Microbiol. Mol. Biol. Rev. 77, 277–321 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Paiva, P., Sousa, S. F., Ramos, M. J. & Fernandes, P. A. Understanding the catalytic machinery and the reaction pathway of the malonyl-acetyl transferase domain of human fatty acid synthase. ACS Catal. 8, 4860–4872 (2018).

    Article  CAS  Google Scholar 

  34. Trent, M. S., Worsham, L. M. S. & Ernst-Fonberg, M. L. HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis. Biochemistry 38, 9541–9548 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Dodds, A. W., Ren, X. D., Willis, A. C. & Law, S. K. The reaction mechanism of the internal thioester in the human complement component C4. Nature 379, 177–179 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Gadjeva, M. et al. The covalent binding reaction of complement component C3. J. Immunol. 161, 985–990 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Elliott, R. J., Bennet, A. J., Braun, C. A., MacLeod, A. M. & Borgford, T. J. Active-site variants of Streptomyces griseus protease B with peptide-ligation activity. Chem. Biol. 7, 163–171 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Bolon, D. N. & Mayo, S. L. Enzyme-like proteins by computational design. Proc. Natl Acad. Sci. USA 98, 14274–14279 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Burke, A. J. et al. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism. Nature 570, 219–223 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Naganuma, T. & Kihara, A. Two modes of regulation of the fatty acid elongase ELOVL6 by the 3-ketoacyl-CoA reductase KAR in the fatty acid elongation cycle. PLoS ONE 9, e101823 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Ghanevati, M. & Jaworski, J. G. Active-site residues of a plant membrane-bound fatty acid elongase beta-ketoacyl-CoA synthase, FAE1 KCS. Biochim. Biophys. Acta 1530, 77–85 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Ghanevati, M. & Jaworski, J. G. Engineering and mechanistic studies of the Arabidopsis FAE1 β-ketoacyl-CoA synthase, FAE1 KCS. Eur. J. Biochem. 269, 3531–3539 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Aldahmesh, M. A. et al. Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia. Am. J. Hum. Genet. 89, 745–750 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Giroux, J. M. & Barbeau, A. Erythrokeratodermia with ataxia. Arch. Dermatol. 106, 183–188 (1972).

    Article  CAS  PubMed  Google Scholar 

  45. Herron, B. J., Bryda, E. C., Heverly, S. A., Collins, D. N. & Flaherty, L. Scraggly, a new hair loss mutation on mouse chromosome 19. Mamm. Genome 10, 864–869 (1999).

    Article  CAS  PubMed  Google Scholar 

  46. Westerberg, R. et al. Role for ELOVL3 and fatty acid chain length in development of hair and skin function. J. Biol. Chem. 279, 5621–5629 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Engelen, M., Kemp, S. & Poll-The, B.-T. X-linked adrenoleukodystrophy: pathogenesis and treatment. Curr. Neurol. Neurosci. Rep. 14, 486 (2014).

    Article  PubMed  Google Scholar 

  48. Sassa, T., Wakashima, T., Ohno, Y. & Kihara, A. Lorenzo’s oil inhibits ELOVL1 and lowers the level of sphingomyelin with a saturated very long-chain fatty acid. J. Lipid Res. 55, 524–530 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Glaser, F. et al. ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics 19, 163–164 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Winter, G. et al. DIALS: implementation and evaluation of a new integration package. Acta Crystallogr. D Biol. Crystallogr. 74, 85–97 (2018).

    Article  CAS  Google Scholar 

  51. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  PubMed  Google Scholar 

  53. Tickle, I. J. et al. STARANISO (Global Phasing Ltd, 2018).

    Google Scholar 

  54. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Terwilliger, T. SOLVE and RESOLVE: automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49–52 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Cowtan, K. D., Zhang, K. Y. J. & Main, P. in International Tables for Crystallography Vol. F (eds. Rossmann, M. G. & Arnold, E.) Ch. 15.3 (International Union of Crystallography, 2012).

  57. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    Article  PubMed  Google Scholar 

  58. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Bricogne, G. et al. BUSTER version 2.10.3 (Global Phasing Ltd, 2017).

    Google Scholar 

  60. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D Biol. Crystallogr. 68, 368–380 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

L.N., A.C.W.P., G.F.R., A.C., V.C., S.R.B., D.S., T.M., L.S., S.M.M.M., N.A.B.-B. and E.P.C. were members of the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, Janssen, Merck KGaA, Merck & Co., Novartis, the Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP and Takeda, as well as the Innovative Medicines Initiative Joint Undertaking ULTRA-DD grant no. 115766 and the Wellcome grant no. 106169/Z/14/Z. T.C.P. is supported by a Wellcome PhD studentship (102164/B/15/Z). A.Q. is supported by Wellcome grant no. 202892/Z/16/Z. We thank Diamond Light Source for beam time (BAG proposal mx19301) and the I24 beamline staff for assistance with beam time, crystal screening and data collection. We thank O. Smart and C. Vonrhein at Global Phasing Ltd for assistance with generating covalent ligand restraints. We acknowledge the use of the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). We are grateful to the Membrane Protein Laboratory (Wellcome grant no. 202892/Z/16/Z) at the Research Complex at Harwell for access to SEC–MALS equipment and assistance with these experiments.

Author information

Author notes

  1. Laiyin Nie

    Present address: CRELUX GmbH, Planegg-Martinsried, Germany

  2. Simon R. Bushell

    Present address: Orbit Discovery, Oxford, UK

  3. Gian Filippo Ruda

    Present address: Evotec Ltd, Milton, Abingdon, UK

  4. Amy Chu

    Present address: Department of Biochemistry, Oxford University, Oxford, UK

  5. Victoria Cole

    Present address: Genomics Centre, South African Medical Research Council, Cape Town, South Africa

  6. James D. Love

    Present address: Novo Nordisk A/S, Måløv, Denmark

  7. Elisabeth P. Carpenter

    Present address: Vertex Pharmaceuticals Ltd, Milton, Abingdon, UK

  8. These authors contributed equally: Laiyin Nie, Tomas C. Pascoa, Ashley C. W. Pike.

Authors and Affiliations

  1. Structural Genomics Consortium, Centre for Medicines Discovery, University of Oxford, Oxford, UK

    Laiyin Nie, Tomas C. Pascoa, Ashley C. W. Pike, Simon R. Bushell, Gian Filippo Ruda, Amy Chu, Victoria Cole, David Speedman, Tiago Moreira, Leela Shrestha, Shubhashish M. M. Mukhopadhyay, Nicola A. Burgess-Brown, Paul E. Brennan & Elisabeth P. Carpenter

  2. Membrane Protein Laboratory, Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, UK

    Andrew Quigley

  3. Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, UK

    Andrew Quigley

  4. Albert Einstein College of Medicine, Department of Biochemistry, Bronx, NY, USA

    James D. Love

  5. Alzheimer’s Research UK Oxford Drug Discovery Institute, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK

    Paul E. Brennan

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Contributions

L.N. purified protein and carried out biophysical characterizations, assisted by V.C. and D.S., and obtained crystals that diffracted to beyond 4-Å resolution. L.N. and A.C.W.P. collected X-ray diffraction data and solved and built the structure. T.C.P. purified protein samples, performed mass spectrometry adduct analysis and SEC–MALS experiments. A.Q. provided access to equipment and assisted in SEC–MALS. G.F.R. assisted with design and execution of the rapid fire mass spectrometry activity assay, supervised by P.E.B. T.M. assisted with mass spectrometry methods optimization. S.R.B. and A.C. were involved in the early stages of the project, including design of constructs, optimization of the protein purification, production and screening of initial crystals and initial mass spectrometry studies. J.D.L. assisted with early stages of the project, including testing constructs and providing materials. Constructs were screened for expression by L.S., and large scale insect cell expressions were produced by S.M.M.M., supervised by N.A.B.B. Data were analyzed and the paper was written by L.N., T.C.P., A.C.W.P., G.F.R., P.E.B. and E.P.C. E.P.C. supervised all aspects of the project.

Corresponding author

Correspondence to Elisabeth P. Carpenter.

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Peer review information Nature Structural & Molecular Biology thanks Lei Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Florian Ullrich 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 Properties of purified ELOVL7.

a, SDS-PAGE gel of purified ELOVL7. Data shown are from a single purification but similar results were observed for multiple purifications / modifications. b, SEC profile and MALS analysis showing that OGNG-solubilised protein exists as a dimer in solution. Experiment carried out for tag-cleaved ELOVL7 purifications with and without IAM modification. Similar results were seen for each sample. c, Representation of head-to-tail dimer present in the crystal. d, ELOVL7 head-to-tail dimer packing within the crystal lattice. e, f, Intact mass analysis of ELOVL7 protein at various stages during purification. Deconvoluted mass spectra are shown for ELOVL7 protein purified from (e) Sf9 (n = 3 or 4) and (f) Expi293F cells (n = 2). For protein purified after expression in insect cells, the samples are shown after immobilized metal affinity chromatography (IMAC), after cleavage of the tag and size exclusion chromatography (SEC) and after treatment with iodoacetamide (IAM). The expected mass of the untagged ‘native’ enzyme (E) based on the sequence is 34222.38 Da. The observed mass peaks (Sf9, 34132.78 Da; Expi 34133.36 Da) correspond to the loss of the N-terminal methionine (−131.20 Da) and acetylation of the resulting new N-terminus (+42.04 Da). All samples were run in their reduced state. The modified material (E*) appears as an adduct with an average mass shift of +1073.6 Da. The addition of 113.55 Da upon treatment with IAM suggests modification of two cysteine residues. g-i, Deconvoluted intact mass spectra for the untagged g, WT (n = 3), h, H150A (n = 2) and i, H181A mutants (n = 2). The expected mass decrease of a His-to-Ala mutation is 66.06 Da. Evidence of in vivo modification (E*) is observed for the H181A mutant but not for H150A. For intact mass experiments, theoretical and experimental masses along with mass errors are given in Supplementary Table 1.

Extended Data Fig. 2 TM helix topology of ELOVL7.

TM helical topology of ELOVL7 is compared with other six and seven membered TM bundles. TM helices are numbered and location of substrate/ligand site marked. Underlying cartoon representations of each structure are colored from blue to red from the N- to C-termini respectively. PDB accession codes are shown in parentheses.

Extended Data Fig. 3 Electron density clearly shows covalently bound 3-keto-eicosanoyl-CoA.

a-d, Electron density running along the catalytic tunnel. Final BUSTER 2mFo-DFc (blue mesh, contoured at 1σ) and omit mFo-DFc (green mesh, contoured at 2.5σ) electron density maps are overlaid on the final model. e, Comparison of a test refinement in which the imidazole groups of H150 and H181 were removed from the model (grey carbon protein atoms / palecyan ligand carbon atoms) and the final model (palecyan protein carbons / violet ligand carbon atoms). The BUSTER 2mFo-DFc (blue mesh, contoured at 1σ) and mFo-DFc (green mesh, contoured at 3σ) maps for the refined histidine-truncated model / unlinked acyl-CoA are shown. f, Comparison of various refined models (green carbons - protein only; palecyan - protein without H150/181 sidechain plus ligand; grey/violet - final model with covalently attached ligand).

Extended Data Fig. 4 Sequence alignment and active site conservation of human ELOVL family members.

a, Sequence alignment of human ELOVL1-7. The conserved histidine box (147HxxHH151) is highlighted by a blue box. Filled circles below alignment indicate residues with a proposed catalytic role (blue) and residues interacting with either the CoA (orange) or acyl (plum) portion of the substrate. Cysteines that form the disulfide bridge (C99-C231) between the TM2/3 and TM6/7 loops are indicated by stars. b, Conservation of active site tunnel. Molecular surface representation is coloured by amino acid conservation score calculated by CONSURF analysis60 of a diverse set of ELOVL1-7 family members. The various subregions of the tunnel are indicated (ADP / Pan from CoA and Acyl chain). Amino acid residues that form the binding tunnel are coloured according to region (pink, acyl; blue, catalytic site; orange CoA binding).

Extended Data Fig. 5 WT ELOVL7 activity.

a,b Activity of residual WT enzyme on incubation with stearoyl-CoA (C18:0) and malonyl-CoA. Selected ion recording is shown for a, reaction mixture without added enzyme and b, reaction mixture after 3hr incubation with ELOVL7 enzyme. The ion peak at 1.61 mins corresponds to the expected 3-keto-eicosanoyl (C20)-CoA product of the elongation reaction. This experiment was carried out with two biological repeats with similar results.

Extended Data Fig. 6 Identification of a covalent acyl-enzyme intermediate of ELOVL7.

Purified, tagged, wild-type ELOVL7 was incubated in the presence and absence of known substrates and metal-chelating agents prior to LC-ESI-MS intact mass analysis. a-g, Deconvoluted intact mass spectra for ELOVL7 incubated for 2h at 37 °C. a, in the absence of substrates. b, ELOVL7 incubated with 100 μM C18:0-CoA. Expected mass addition for acyl intermediate upon reaction with C18:0-CoA: +266.47 Da. c, ELOVL7 incubated with 100μM C18:3(n3)-CoA. Expected mass addition for acyl intermediate upon reaction with C18:3(n3)-CoA: +260.42 Da. d-e, ELOVL7 incubated with d, 100 μM C18:0-CoA or e, 100 μM C18:3(n3)-CoA in the presence of 1mM EDTA. f-g ELOVL7 incubated with f, 100 μM C18:0-CoA or g, 100 μM C18:3(n3)-CoA in the presence of 1mM EGTA. h-k, Sequential reaction of LMNG-purified ELOVL7 with C18:0-CoA and malonyl-CoA. h, LMNG-purified ELOVL in the absence of substrates. i, ELOVL7 incubated with 100 μM C18:0-CoA. j, Purified ELOVL7 initially incubated with 100 μM C18:0-CoA, followed by incubation with 200 μM malonyl-CoA. Addition of the second substrate leads to loss of the acyl-enzyme intermediate peak, consistent with the reaction having gone to completion. k, control ELOVL7 sample taken after incubation with C18:0-CoA was further incubated in the absence of malonyl-CoA, showing that covalent intermediate loss only occurs in the presence of malonyl-CoA. All experiments were repeated independently twice with similar results (n = 2 biological repeats, see Supplementary Figs. 1 and 2 for replicate traces. See Supplementary Table 2 for theoretical and experimental masses and mass errors).

Extended Data Fig. 7 Proposed ping-pong reaction mechanism for ELOVL7.

a, Transacylation step with acyl chain of the first substrate being transferred to H150. In the second step, malonyl-CoA binds and undergoes decarboxylation and a condensation reaction to form the elongated 3-keto product. b, Proposed reaction steps leading to C-N covalent adduct with H150 seen in crystal structure. In this scenario a 2,3-trans-enoyl-CoA serves as the first substrate (left) leading to the 3-keto,4,5-trans-enoyl-CoA ‘product’ (middle) which subsequently crosslinks to H150 via a conjugate addition reaction of H150 (right). The nature of the reaction that leads to H181 crosslinking to the C2 atom of the 3-keto-acyl-CoA is not clear.

Extended Data Table 1 Mutagenesis primer sequences
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Nie, L., Pascoa, T.C., Pike, A.C.W. et al. The structural basis of fatty acid elongation by the ELOVL elongases. Nat Struct Mol Biol 28, 512–520 (2021). https://doi.org/10.1038/s41594-021-00605-6

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