Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Architecture and functional dynamics of the pentafunctional AROM complex

Abstract

The AROM complex is a multifunctional metabolic machine with ten enzymatic domains catalyzing the five central steps of the shikimate pathway in fungi and protists. We determined its crystal structure and catalytic behavior, and elucidated its conformational space using a combination of experimental and computational approaches. We derived this space in an elementary approach, exploiting an abundance of conformational information from its monofunctional homologs in the Protein Data Bank. It demonstrates how AROM is optimized for spatial compactness while allowing for unrestricted conformational transitions and a decoupled functioning of its individual enzymatic entities. With this architecture, AROM poses a tractable test case for the effects of active site proximity on the efficiency of both natural metabolic systems and biotechnological pathway optimization approaches. We show that a mere colocalization of enzymes is not sufficient to yield a detectable improvement of metabolic throughput.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Catalytic steps of the SKM pathway.
Fig. 2: The architecture and structural characteristics of the AROM complex.
Fig. 3: Enzymatic activity of the AROM complex.
Fig. 4: Conformational space, SAXS and crosslinking analysis of the AROM complex.

Similar content being viewed by others

Data availability

The coordinates and structural factors of the crystal structure have been deposited in the PDB under accession code 6HQV, and the SAXS data in the Small Angle Scattering Biological Data Bank55 under accession code SASDHP8. Mass spectrometry data have been deposited to the ProteomeXchange Consortium56 via the PRIDE partner repository with the dataset identifier PXD010479. All other relevant data are available in this article and its supplementary information files, or from the corresponding author upon reasonable request.

References

  1. Duke, S. O. & Powles, S. B. Glyphosate: a once-in-a-century herbicide. Pest Manag. Sci. 64, 319–325 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Bentley, R. The shikimate pathway—a metabolic tree with many branches. Crit. Rev. Biochem. Mol. Biol. 25, 307–384 (1990).

    Article  CAS  PubMed  Google Scholar 

  3. Ahmed, S. I. & Giles, N. H. Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J. Bacteriol. 99, 231–237 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Roberts, C. W. et al. The shikimate pathway and its branches in apicomplexan parasites. J. Infect. Dis. 185, S25–S36 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Richards, T. A. et al. Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements. Eukaryot. Cell 5, 1517–1531 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gourley, D. G. et al. The two types of 3-dehydroquinase have distinct structures but catalyze the same overall reaction. Nat. Struct. Biol. 6, 521–525 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Carpenter, E. P., Hawkins, A. R., Frost, J. W. & Brown, K. A. Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis. Nature 394, 299–302 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Schonbrunn, E. et al. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proc. Natl Acad. Sci. USA 98, 1376–1380 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hartmann, M. D., Bourenkov, G. P., Oberschall, A., Strizhov, N. & Bartunik, H. D. Mechanism of phosphoryl transfer catalyzed by shikimate kinase from Mycobacterium tuberculosis. J. Mol. Biol. 364, 411–423 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Michel, G. et al. Structures of shikimate dehydrogenase AroE and its paralog YdiB—a common structural framework for different activities. J. Biol. Chem. 278, 19463–19472 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Derrer, B., Macheroux, P. & Kappes, B. The shikimate pathway in apicomplexan parasites: implications for drug development. Front. Biosci. (Landmark Ed.) 18, 944–969 (2013).

    Article  CAS  Google Scholar 

  12. Peek, J., Castiglione, G., Shi, T. & Christendat, D. Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex. Mol. Biochem. Parasitol. 194, 16–19 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Campbell, S. A. et al. A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation. Int. J. Parasitol. 34, 5–13 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Dunn, M. F. Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex. Arch. Biochem. Biophys. 519, 154–166 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maier, T., Leibundgut, M., Boehringer, D. & Ban, N. Structure and function of eukaryotic fatty acid synthases. Q. Rev. Biophys. 43, 373–422 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Keatinge-Clay, A. T. The structures of type I polyketide synthases. Nat. Prod. Rep. 29, 1050–1073 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Herbst, D. A., Townsend, C. A. & Maier, T. The architectures of iterative type I PKS and FAS. Nat. Prod. Rep. 35, 1046–1069 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bernhardsgrutter, I. et al. The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite. Nat. Chem. Biol. 14, 1127–1132 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Conrado, R. J., Varner, J. D. & DeLisa, M. P. Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr. Opin. Biotech. 19, 492–499 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Schmid-Dannert, C. & Lopez-Gallego, F. Advances and opportunities for the design of self-sufficient and spatially organized cell-free biocatalytic systems. Curr. Opin. Chem. Biol. 49, 97–104 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Jia, F., Narasimhan, B. & Mallapragada, S. Materials-based strategies for multi-enzyme immobilization and co-localization: a review. Biotechnol. Bioeng. 111, 209–222 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Burley, S. K. et al. RCSB Protein Data Bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy. Nucleic Acids Res. 47, D464–D474 (2019).

    Article  CAS  PubMed  Google Scholar 

  23. Leitner, A. et al. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes. Proc. Natl Acad. Sci. USA 111, 9455–9460 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Henriquez, F. L. et al. The Acanthamoeba shikimate pathway has a unique molecular arrangement and is essential for aromatic amino acid biosynthesis. Protist 166, 93–105 (2015).

    Article  CAS  PubMed  Google Scholar 

  25. Yeoh, L. M., Lee, V. V., McFadden, G. I. & Ralph, S. A. Alternative splicing in apicomplexan parasites. mBio 10, https://doi.org/10.1128/mBio.02866-18 (2019).

  26. Lamb, H. K. et al. Comparative analysis of the QUTR transcription repressor protein and the three C-terminal domains of the pentafunctional AROM enzyme. Biochem. J. 313, 941–950 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Levett, L. J. et al. Identification of domains responsible for signal recognition and transduction within the QUTR transcription repressor protein. Biochem. J. 350(Pt 1), 189–197 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ding, S. W., Cargill, A. A., Medintz, I. L. & Claussen, J. C. Increasing the activity of immobilized enzymes with nanoparticle conjugation. Curr. Opin. Biotech. 34, 242–250 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Zhang, Y. F., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade. Nat. Commun. 7, 13982 (2016).

  30. Shatalin, K., Lebreton, S., Rault-Leonardon, M., Velot, 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).

    Article  CAS  PubMed  Google Scholar 

  31. Lin, Y., Boese, C. J. & St Maurice, M. The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent. Protein Sci. 25, 1812–1824 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bauerle, R., Hess, J. & French, S. Anthranilate synthase–anthranilate phosphoribosyltransferase complex and subunits of Salmonella typhimurium. Methods Enzymol. 142, 366–386 (1987).

    Article  CAS  PubMed  Google Scholar 

  33. Doublié, S. in Macromolecular Crystallography Protocols: Volume 1, Preparation and Crystallization of Macromolecules (eds Walker, J. M. & Doublié, S.) 91–108 (Humana Press, 2007).

  34. Sivashanmugam, A. et al. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci. 18, 936–948 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tutino, M. L., Tosco, A., Marino, G. & Sannia, G. Expression of Sulfolobus solfataricus trpE and trpG genes in E. coli. Biochem. Biophys. Res. Commun. 230, 306–310 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. 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 

  40. Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D Biol. Crystallogr. 55, 247–255 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Forster, S., Apostol, L. & Bras, W. Scatter: software for the analysis of nano- and mesoscale small-angle scattering. J. Appl. Crystallogr. 43, 639–646 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  46. Schneidman-Duhovny, D., Hammel, M., Tainer, J. A. & Sali, A. Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophys. J. 105, 962–974 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Use R!) (Springer, 2009).

  48. Leitner, A., Walzthoeni, T. & Aebersold, R. Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline. Nat. Protoc. 9, 120–137 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Walzthoeni, T. et al. False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nat. Methods 9, 901–903 (2012).

    Article  CAS  PubMed  Google Scholar 

  50. van Zundert, G. C. & Bonvin, A. M. DisVis: quantifying and visualizing accessible interaction space of distance-restrained biomolecular complexes. Bioinformatics 31, 3222–3224 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. van Zundert, G. C. et al. The DisVis and PowerFit web servers: explorative and integrative modeling of biomolecular complexes. J. Mol. Biol. 429, 399–407 (2017).

    Article  PubMed  CAS  Google Scholar 

  52. Zimmermann, L. et al. A completely reimplemented MPI Bioinformatics Toolkit with a new HHpred server at its core. J. Mol. Biol. 430, 2237–2243 (2018).

    Article  CAS  PubMed  Google Scholar 

  53. Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 54, 5.6.1–5.6.37 (2016).

    Article  Google Scholar 

  55. Valentini, E., Kikhney, A. G., Previtali, G., Jeffries, C. M. & Svergun, D. I. SASBDB, a repository for biological small-angle scattering data. Nucleic Acids Res. 43, D357–D363 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Deutsch, E. W. et al. The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition. Nucleic Acids Res. 45, D1100–d1106 (2017).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Lupas for continuous support; M. Flötenmeyer for performing electron microscopy; A. Ursinus, S. Grüner, C. Heim and E. Valkov for experimental assistance and advice; and R. Aebersold for access to infrastructure and instrumentation for XL-MS experiments. We thank Diamond Light Source for access to the SAXS beamline B21 (proposal SM14307) that contributed to the results presented here, and thank R. Rambo and N. Cowieson for assistance in using the beamline. Crystallographic data were collected at beamline P14 operated by EMBL Hamburg at the PETRAIII storage ring (DESY, Hamburg, Germany). We thank G. Bourenkov for the assistance in using the beamline. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (grant agreement No. 283570), and was supported by institutional funds from the Max Planck Society.

Author information

Authors and Affiliations

Authors

Contributions

H.A.V. initiated the study, designed constructs and performed expression, protein purification, circular dichroism spectroscopy, multi-angle light scattering, kinetics, crystallography and SAXS experiments. M.L. established the high-cell-density expression protocol and performed expression, protein purification and kinetics experiments. R.A. designed constructs and performed cloning, expression, protein purification and dimerization dynamics experiments and crystallography. A.L. performed XL-MS. H.Z. performed conformational analysis and homology modeling, produced Supplementary Video 1 and contributed to the analysis of kinetic data. M.D.H. conceived and supervised the study, performed kinetics, crystallography and conformational analysis, and wrote the paper with contributions from all other authors.

Corresponding author

Correspondence to Marcus D. Hartmann.

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 Information

Supplementary Figs. 1–7 and Table 1.

Reporting Summary

Supplementary Data 1

Excel sheet containing all identified crosslinks.

Supplementary Video 1

Video illustrating the conformational space, indicating how the constituent AROM domains can undergo conformational changes without the need of coordination.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Arora Verasztó, H., Logotheti, M., Albrecht, R. et al. Architecture and functional dynamics of the pentafunctional AROM complex. Nat Chem Biol 16, 973–978 (2020). https://doi.org/10.1038/s41589-020-0587-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0587-9

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing