Skip to main content

Thank you for visiting 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.

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


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.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


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

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.

Accession codes

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).


  1. Berg, I. A. et al. Autotrophic carbon fixation in archaea. Nature Rev. Microbiol. 8, 447–460 (2010)

    Article  CAS  Google Scholar 

  2. Say, R. F. & Fuchs, G. Fructose 1,6-bisphosphate aldolase/phosphatase may be an ancestral gluconeogenic enzyme. Nature 464, 1077–1081 (2010)

    Article  ADS  CAS  Google Scholar 

  3. Stetter, K. O. Hyperthermophiles in the history of life. Phil. Trans. R. Soc. B 361, 1837–1842 (2006)

    Article  CAS  Google Scholar 

  4. Siebers, B. et al. Archaeal fructose-1,6-bisphosphate aldolases constitute a new family of archaeal type class I aldolase. J. Biol. Chem. 276, 28710–28718 (2001)

    Article  CAS  Google Scholar 

  5. Messner, P., Pum, D., Sara, M., Stetter, K. O. & Sleytr, U. B. Ultrastructure of the cell envelope of the archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus. J. Bacteriol. 166, 1046–1054 (1986)

    Article  CAS  Google Scholar 

  6. Zillig, W. et al. The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria. Can. J. Microbiol. 35, 73–80 (1989)

    Article  CAS  Google Scholar 

  7. Alefounder, P. R., Baldwin, S. A., Perham, R. N. & Short, N. J. Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli. Biochem. J. 257, 529–534 (1989)

    Article  CAS  Google Scholar 

  8. Fothergill-Gilmore, L. A. & Michels, P. A. Evolution of glycolysis. Prog. Biophys. Mol. Biol. 59, 105–235 (1993)

    Article  CAS  Google Scholar 

  9. Rutter, W. J. Evolution of aldolase. Fed. Proc. 23, 1248–1257 (1964)

    CAS  PubMed  Google Scholar 

  10. Lorentzen, E., Siebers, B., Hensel, R. & Pohl, E. Structure, function and evolution of the archaeal class I fructose-1,6-bisphosphate aldolase. Biochem. Soc. Trans. 32, 259–263 (2004)

    Article  CAS  Google Scholar 

  11. Penhoet, E., Kochman, M., Valentine, R. & Rutter, W. J. The subunit structure of mammalian fructose diphosphate aldolase. Biochemistry 6, 2940–2949 (1967)

    Article  CAS  Google Scholar 

  12. Tolan, D. R., Niclas, J., Bruce, B. D. & Lebo, R. V. Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations. Am. J. Hum. Genet. 41, 907–924 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Lebherz, H. G. & Rutter, W. J. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 8, 109–121 (1969)

    Article  CAS  Google Scholar 

  14. Cooper, S. J. et al. The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold. Structure 4, 1303–1315 (1996)

    Article  CAS  Google Scholar 

  15. Lorentzen, E. et al. Crystal structure of an archaeal class I aldolase and the evolution of (βα)8 barrel proteins. J. Biol. Chem. 278, 47253–47260 (2003)

    Article  CAS  Google Scholar 

  16. Hester, G. et al. The crystal structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster at 2.5 Å resolution. FEBS Lett. 292, 237–242 (1991)

    Article  CAS  Google Scholar 

  17. Gamblin, S. J. et al. The crystal structure of human muscle aldolase at 3.0 Å resolution. FEBS Lett. 262, 282–286 (1990)

    Article  CAS  Google Scholar 

  18. Blom, N. S., Tetreault, S., Coulombe, R. & Sygusch, J. Novel active site in Escherichia coli fructose-1,6-bisphosphate aldolase. Nature Struct. Biol. 3, 856–862 (1996)

    Article  CAS  Google Scholar 

  19. Imanaka, H., Fukui, T., Atomi, H. & Imanaka, T. Gene cloning and characterization of fructose-1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Biosci. Bioeng. 94, 237–243 (2002)

    Article  CAS  Google Scholar 

  20. Andreeva, A. et al. Data growth and its impact on the SCOP database: new developments. Nucleic Acids Res. 36, 419–425 (2008)

    Article  Google Scholar 

  21. Ronimus, R. S. & Morgan, H. W. Distribution and phylogenies of enzymes of the Embden-Meyerhof-Parnas pathway from archaea and hyperthermophilic bacteria support a gluconeogenic origin of metabolism. Archaea 1, 199–221 (2003)

    Article  CAS  Google Scholar 

  22. Nishimasu, H., Fushinobu, S., Shoun, H. & Wakagi, T. The first crystal structure of the novel class of fructose-1,6-bisphosphatase present in thermophilic archaea. Structure 12, 949–959 (2004)

    Article  CAS  Google Scholar 

  23. St.-Jean, M., Blonski, C. & Sygusch, J. Charge stabilization and entropy reduction of central lysine residues in fructose-bisphosphate aldolase. Biochemistry 48, 4528–4537 (2009)

    Article  CAS  Google Scholar 

  24. Grazi, E., Rowley, P. T., Cheng, T., Tchola, O. & Horecker, B. L. The mechanism of action of aldolases. III. Schiff base formation with lysine. Biochem. Biophys. Res. Commun. 9, 38–43 (1962)

    Article  CAS  Google Scholar 

  25. Rose, I. A. & Rieder, S. V. Studies on the mechanism on the aldolase reaction; isotope exchange reactions of muscle and yeast aldolase. J. Biol. Chem. 231, 315–329 (1958)

    CAS  PubMed  Google Scholar 

  26. Lai, C. Y., Tchola, O., Cheng, T. & Horecker, B. L. The mechanism of action of aldolases. 8. The number of combining sites in fructose diphosphate aldolase. J. Biol. Chem. 240, 1347–1350 (1965)

    CAS  PubMed  Google Scholar 

  27. St.-Jean, M., Lafrance-Vanasse, J., Liotard, B. & Sygusch, J. High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit. J. Biol. Chem. 280, 27262–27270 (2005)

    Article  CAS  Google Scholar 

  28. Ciszak, E. M., Korotchkina, L. G., Dominiak, P. M., Sidhu, S. & Patel, M. S. Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase. J. Biol. Chem. 278, 21240–21246 (2003)

    Article  CAS  Google Scholar 

  29. Perham, R. N. Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. Annu. Rev. Biochem. 69, 961–1004 (2000)

    Article  CAS  Google Scholar 

  30. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26. (1992)

  31. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  32. Vagin, A. A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997)

    Article  CAS  Google Scholar 

  33. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  35. Brünger, A. T. Assessment of phase accuracy by cross validation - the free R-value – methods and applications. Acta Crystallogr. D 49, 24–36 (1993)

    Article  Google Scholar 

  36. Tabor, S. & Richardson, C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl Acad. Sci. USA 82, 1074–1078 (1985)

    Article  ADS  CAS  Google Scholar 

  37. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)

    Article  CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Oliver Einsle.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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