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
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
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
Duke, S. O. & Powles, S. B. Glyphosate: a once-in-a-century herbicide. Pest Manag. Sci. 64, 319–325 (2008).
Bentley, R. The shikimate pathway—a metabolic tree with many branches. Crit. Rev. Biochem. Mol. Biol. 25, 307–384 (1990).
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).
Roberts, C. W. et al. The shikimate pathway and its branches in apicomplexan parasites. J. Infect. Dis. 185, S25–S36 (2002).
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).
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).
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).
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).
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).
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).
Derrer, B., Macheroux, P. & Kappes, B. The shikimate pathway in apicomplexan parasites: implications for drug development. Front. Biosci. (Landmark Ed.) 18, 944–969 (2013).
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).
Campbell, S. A. et al. A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation. Int. J. Parasitol. 34, 5–13 (2004).
Dunn, M. F. Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex. Arch. Biochem. Biophys. 519, 154–166 (2012).
Maier, T., Leibundgut, M., Boehringer, D. & Ban, N. Structure and function of eukaryotic fatty acid synthases. Q. Rev. Biophys. 43, 373–422 (2010).
Keatinge-Clay, A. T. The structures of type I polyketide synthases. Nat. Prod. Rep. 29, 1050–1073 (2012).
Herbst, D. A., Townsend, C. A. & Maier, T. The architectures of iterative type I PKS and FAS. Nat. Prod. Rep. 35, 1046–1069 (2018).
Bernhardsgrutter, I. et al. The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite. Nat. Chem. Biol. 14, 1127–1132 (2018).
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).
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).
Jia, F., Narasimhan, B. & Mallapragada, S. Materials-based strategies for multi-enzyme immobilization and co-localization: a review. Biotechnol. Bioeng. 111, 209–222 (2014).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Bauerle, R., Hess, J. & French, S. Anthranilate synthase–anthranilate phosphoribosyltransferase complex and subunits of Salmonella typhimurium. Methods Enzymol. 142, 366–386 (1987).
Doublié, S. in Macromolecular Crystallography Protocols: Volume 1, Preparation and Crystallization of Macromolecules (eds Walker, J. M. & Doublié, S.) 91–108 (Humana Press, 2007).
Sivashanmugam, A. et al. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci. 18, 936–948 (2009).
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).
Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).
Sheldrick, G. A short history of SHELX. Acta Crystallogr. A Found. Crystallogr. 64, 112–122 (2008).
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Struct. Biol. 62, 1002–1011 (2006).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
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).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285 (2016).
Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).
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).
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).
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).
Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Use R!) (Springer, 2009).
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).
Walzthoeni, T. et al. False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nat. Methods 9, 901–903 (2012).
van Zundert, G. C. & Bonvin, A. M. DisVis: quantifying and visualizing accessible interaction space of distance-restrained biomolecular complexes. Bioinformatics 31, 3222–3224 (2015).
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).
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).
Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008).
Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 54, 5.6.1–5.6.37 (2016).
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).
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).
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
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
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.
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
About this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41589-020-0587-9
This article is cited by
-
Channeling a complex question
Nature Chemical Biology (2020)