The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite


Cells must cope with toxic or reactive intermediates formed during metabolism. One coping strategy is to sequester reactions that produce such intermediates within specialized compartments or tunnels connecting different active sites. Here, we show that propionyl-CoA synthase (PCS), an  400-kDa homodimer, three-domain fusion protein and the key enzyme of the 3-hydroxypropionate bi-cycle for CO2 fixation, sequesters its reactive intermediate acrylyl-CoA. Structural analysis showed that PCS forms a multicatalytic reaction chamber. Kinetic analysis suggested that access to the reaction chamber and catalysis are synchronized by interdomain communication. The reaction chamber of PCS features three active sites and has a volume of only 33 nm3. As one of the smallest multireaction chambers described in biology, PCS may inspire the engineering of a new class of dynamically regulated nanoreactors.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Trifunctional PCS: structure and reaction sequence.
Fig. 2: PCS sequesters the reactive intermediate acrylyl-CoA.
Fig. 3: Multicatalytic reaction chamber of PCS.
Fig. 4: Proposed catalytic cycle of PCS.

Data availability

The coordinates and structure factors of the crystal structure generated from this research are available in the Protein Data Bank under accession number PDB 6EQO. All other relevant data are available in this article and its supplementary information files, or from the corresponding author upon reasonable request.


  1. 1.

    Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309 (2016).

  2. 2.

    Linster, C. L., Van Schaftingen, E. & Hanson, A. D. Metabolite damage and its repair or pre-emption. Nat. Chem. Biol. 9, 72–80 (2013).

  3. 3.

    Alber, B. E. & Fuchs, G. Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation. J. Biol. Chem. 277, 12137–12143 (2002).

  4. 4.

    Zarzycki, J., Brecht, V., Müller, M. & Fuchs, G. Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus. Proc. Natl Acad. Sci. USA 106, 21317–21322 (2009).

  5. 5.

    Todd, J. D., Curson, A. R. J., Sullivan, M. J., Kirkwood, M. & Johnston, A. W. B. The Ruegeria pomeroyi acuI gene has a role in DMSP catabolism and resembles yhdH of E. coli and other bacteria in conferring resistance to acrylate. PLoS One 7, e35947 (2012).

  6. 6.

    Teufel, R., Kung, J. W., Kockelkorn, D., Alber, B. E. & Fuchs, G. 3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenzyme A reductase, enzymes of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in the Sulfolobales. J. Bacteriol. 191, 4572–4581 (2009).

  7. 7.

    Berg, I. A., Kockelkorn, D., Buckel, W. & Fuchs, G. A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science 318, 1782–1786 (2007).

  8. 8.

    Engilberge, S. et al. Crystallophore: a versatile lanthanide complex for protein crystallography combining nucleating effects, phasing properties, and luminescence. Chem. Sci. 8, 5909–5917 (2017).

  9. 9.

    Lindbladh, C. et al. Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. Biochemistry 33, 11692–11698 (1994).

  10. 10.

    Shatalin, K., Lebreton, S., Rault-Leonardon, M., Vélot, C. & Srere, P. A. Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase. Biochemistry 38, 881–889 (1999).

  11. 11.

    Datta, A., Merz, J. M. & Spivey, H. O. Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase. J. Biol. Chem. 260, 15008–15012 (1985).

  12. 12.

    Chowdhury, C., Sinha, S., Chun, S., Yeates, T. O. & Bobik, T. A. Diverse bacterial microcompartment organelles. Microbiol. Mol. Biol. Rev. 78, 438–468 (2014).

  13. 13.

    Sutter, M., Greber, B., Aussignargues, C. & Kerfeld, C. A. Assembly principles and structure of a 6.5-MDa bacterial microcompartment shell. Science 356, 1293–1297 (2017).

  14. 14.

    Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat. Struct. Mol. Biol. 15, 939–947 (2008).

  15. 15.

    Jung, T. & Grune, T. The proteasome and the degradation of oxidized proteins: Part I—structure of proteasomes. Redox Biol. 1, 178–182 (2013).

  16. 16.

    Reger, A. S., Carney, J. M. & Gulick, A. M. Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase. Biochemistry 46, 6536–6546 (2007).

  17. 17.

    Bock, T., Reichelt, J., Müller, R. & Blankenfeldt, W. The structure of LiuC, a 3-hydroxy-3-methylglutaconyl CoA dehydratase involved in isovaleryl-CoA biosynthesis in Myxococcus xanthus, reveals insights into specificity and catalysis. Chembiochem 17, 1658–1664 (2016).

  18. 18.

    Quade, N., Huo, L., Rachid, S., Heinz, D. W. & Müller, R. Unusual carbon fixation gives rise to diverse polyketide extender units. Nat. Chem. Biol. 8, 117–124 (2011).

  19. 19.

    Spivey, H. O. & Ovádi, J. Substrate channeling. Methods 19, 306–321 (1999).

  20. 20.

    Lyle, S., Ozeran, J. D., Stanczak, J., Westley, J. & Schwartz, N.B. Intermediate channeling between ATP sulfurylase and adenosine 5′-phosphosulfate kinase from rat chondrosarcoma. Biochemistry 33, 6822–6827 (1994).

  21. 21.

    Jogl, G. & Tong, L. Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP. Biochemistry 43, 1425–1431 (2004).

  22. 22.

    Tanaka, S., Sawaya, M. R. & Yeates, T. O. Structure and mechanisms of a protein-based organelle in Escherichia coli. Science 327, 81–84 (2010).

  23. 23.

    Giessen, T. W. & Silver, P. A. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat. Microbiol. 2, 17029 (2017).

  24. 24.

    Fan, C., Cheng, S., Sinha, S. & Bobik, T. A. Interactions between the termini of lumen enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments. Proc. Natl Acad. Sci. USA 109, 14995–15000 (2012).

  25. 25.

    Kerfeld, C. A. et al. Protein structures forming the shell of primitive bacterial organelles. Science 309, 936–938 (2005).

  26. 26.

    Pan, P., Woehl, E. & Dunn, M. F. Protein architecture, dynamics and allostery in tryptophan synthase channeling. Trends. Biochem. Sci. 22, 22–27 (1997).

  27. 27.

    Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W. & Davies, D. R. Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263, 17857–17871 (1988).

  28. 28.

    Mouilleron, S., Badet-Denisot, M.-A. & Golinelli-Pimpaneau, B. Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase. J. Biol. Chem. 281, 4404–4412 (2006).

  29. 29.

    Thoden, J. B., Holden, H. M., Wesenberg, G., Raushel, F. M. & Rayment, I. Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product. Biochemistry 36, 6305–6316 (1997).

  30. 30.

    Singh, H., Arentson, B. W., Becker, D. F. & Tanner, J. J. Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site. Proc. Natl Acad. Sci.USA 111, 3389–3394 (2014).

  31. 31.

    Smith, N. E., Vrielink, A., Attwood, P. V. & Corry, B. Biological channeling of a reactive intermediate in the bifunctional enzyme DmpFG. Biophys. J. 102, 868–877 (2012).

  32. 32.

    Leys, D., Basran, J. & Scrutton, N. S. Channelling and formation of ‘active’ formaldehyde in dimethylglycine oxidase. EMBO J. 22, 4038–4048 (2003).

  33. 33.

    Tralau, T. et al. An internal reaction chamber in dimethylglycine oxidase provides efficient protection from exposure to toxic formaldehyde. J. Biol. Chem. 284, 17826–17834 (2009).

  34. 34.

    Ishikawa, M., Tsuchiya, D., Oyama, T., Tsunaka, Y. & Morikawa, K. Structural basis for channelling mechanism of a fatty acid β-oxidation multienzyme complex. EMBO J. 23, 2745–2754 (2004).

  35. 35.

    Smith, S. & Tsai, S.-C. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat. Prod. Rep. 24, 1041–1072 (2007).

  36. 36.

    Vögeli, B. et al. Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site. Proc. Natl Acad. Sci. USA 115, 3380–3385 (2018).

  37. 37.

    Bale, J. B. et al. Accurate design of megadalton-scale two-component icosahedral protein complexes. Science 353, 389–394 (2016).

  38. 38.

    Aussignargues, C. et al. Structure and function of a bacterial microcompartment shell protein engineered to bind a [4Fe-4S] cluster. J. Am. Chem. Soc. 138, 5262–5270 (2016).

  39. 39.

    Giessen, T. W. & Silver, P. A. A catalytic nanoreactor based on in vivo encapsulation of multiple enzymes in an engineered protein nanocompartment. Chembiochem 17, 1931–1935 (2016).

  40. 40.

    Azuma, Y., Zschoche, R., Tinzl, M. & Hilvert, D. Quantitative packaging of active enzymes into a protein cage. Angew. Chem. Int. Ed. Engl. 55, 1531–1534 (2016).

  41. 41.

    Burton, A. J., Thomson, A. R., Dawson, W. M., Brady, R. L. & Woolfson, D. N. Installing hydrolytic activity into a completely de novo protein framework. Nat. Chem. 8, 837–844 (2016).

  42. 42.

    Brasch, M. et al. Assembling enzymatic cascade pathways inside virus-based nanocages using dual-tasking nucleic acid tags. J. Am. Chem. Soc. 139, 1512–1519 (2017).

  43. 43.

    Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008).

  44. 44.

    Peter, D. M., Vögeli, B., Cortina, N. S. & Erb, T. J. A chemo-enzymatic road map to the synthesis of CoA esters. Molecules 21, 517 (2016).

  45. 45.

    Schwander, T., Schada von Borzyskowski, L., Burgener, S., Cortina, N. S. & Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 354, 900–904 (2016).

  46. 46.

    Bertani, G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic. Escherichia coli. J. Bacteriol. 62, 293–300 (1951).

  47. 47.

    Tartof, K. & Hobbs, C. Improved media for growing plasmid and cosmid clones. Focus 9, 12 (1987).

  48. 48.

    Sambrook, J. & Russel, D. Molecular Cloning: a Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

  49. 49.

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A. 64, 112–122 (2008).

  50. 50.

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

  51. 51.

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

  52. 52.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

  53. 53.

    Eichacker, L., Granvogl, B. & Gruber, P. Method for quantitative comparison of two or more proteins. German patent EP1947461 (2008).

  54. 54.

    Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. & Svergun, D. I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003).

Download references


We thank T. Glatter for conceptualizing the limited proteolysis experiments and P. Pausch for help in collecting SAXS data. We acknowledge support from the European Synchrotron Radiation (ESRF, beamlines ID29, BM30 and BM29), Grenoble, France, and the Synchrotron SOLEIL (beamline PX-2A), Paris, France. The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under contract no. DE-AC02-05CH11231, granted to T.J.E. This work was funded by the Deutsche Forschungsgemeinschaft through Collaborative Research Centre SFB 987, the European Research Council (ERC 637675 ‘SYBORG’), a FET-Open Grant 686330 (‘FutureAgriculture’), the Gebert-Rüf-Stiftung (GRS 062-12) and the Max-Planck-Society, all granted to T.J.E.

Author information

I.B., B.V., D.M.P., J.Z. and T.J.E. conceived the project. I.B., B.V., T.W., J.Z. and T.J.E. designed and performed experiments and analyzed the data. E.G., F.R. and O.M. designed and prepared the phasing compound Tb-Xo4. I.B., B.V., S.E., E.G., T.W. and J.Z. collected X-ray datasets, and T.W. and J.Z. solved crystal structures. E.G., G.B., and S.S oversaw crystallography and SAXS experiments and provided equipment and beam time. J.K. performed peptide-labeling experiments. N.S.C. and J.K. performed mass spectrometry and analyzed the data. I.B., B.V., T.W., J.Z. and T.J.E. wrote the manuscript with contributions from all other authors.

Correspondence to Tobias J. Erb.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–5 and Supplementary Figures 1–17

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bernhardsgrütter, I., Vögeli, B., Wagner, T. et al. The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite. Nat Chem Biol 14, 1127–1132 (2018).

Download citation

Further reading