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.

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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.

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.

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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.

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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.

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Correspondence to Marcus D. Hartmann.

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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.

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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

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