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Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase

Nature volume 478, pages 534–537 (2011)Cite this article

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Abstract

Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages1,2,3. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP4, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus5,6 in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3 Å. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg2+ cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures1,2, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality.

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Figure 1: Representation of the reaction steps of FBP aldolase/phosphatase.
Figure 2: The successive reaction steps of FBP aldolase/phosphatase in the crystal structures.
Figure 3: Catalytic activity of distinct variants of FBP aldolase/phosphatase.
Figure 4: Proposed reaction mechanism of FBP aldolase/phosphatase.

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Data deposits

Atomic coordinates and structure factors for the reported crystal structures are deposited in Protein Data Bank under accession numbers 3T2B (ligand-free), 3T2C (DHAP-bound), 3T2D (FBP-bound), 3T2E (F6P-bound), 3T2F (EDTA-soak with DHAP) and 3T2G (Y229F variant with DHAP).

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Acknowledgements

This work was supported by Deutsche Forschungsgemeinschaft (grant Ei520/3-2 to O.E., Fu118/15-4 and 15-5 to G.F.). Diffraction data were collected at beam lines X06SA and X06DA at the Swiss Light Source (Villigen, Switzerland). The authors thank the beam line staff for assistance during data collection.

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Author notes

  1. Juan Du and Rafael F. Say: These authors contributed equally to this work.

Authors and Affiliations

  1. Lehrstuhl für Biochemie, Institut für organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg, Germany

    Juan Du, Wei Lü & Oliver Einsle

  2. Lehrstuhl für Mikrobiologie, Albert-Ludwigs-Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany

    Rafael F. Say & Georg Fuchs

  3. BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, Hebelstrasse 25, 79104 Freiburg, Germany

    Oliver Einsle

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Contributions

J.D. and W.L. crystallized the protein and collected diffraction data, J.D., W.L. and O.E. built and refined the structural models, R.F.S. created and analysed the variant proteins, J.D., R.F.S., W.L., G.F. and O.E. designed the experiments, O.E. wrote the manuscript.

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Correspondence to Oliver Einsle.

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The authors declare no competing financial interests.

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The file contains Supplementary Figures 1-7 with legends, Supplementary Methods and Supplementary Tables 1-2. (PDF 1366 kb)

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Du, J., Say, R., Lü, W. et al. Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase. Nature 478, 534–537 (2011). https://doi.org/10.1038/nature10458

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