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Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase

Abstract

Enzymes catalyse specific reactions and are essential for maintaining life. Although some are referred to as being bifunctional, they consist of either two distinct catalytic domains or a single domain that displays promiscuous substrate specificity1. Thus, one enzyme active site is generally responsible for one biochemical reaction. In contrast to this conventional concept, archaeal fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) consists of a single catalytic domain, but catalyses two chemically distinct reactions of gluconeogenesis: (1) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP; (2) the dephosphorylation of FBP to fructose-6-phosphate (F6P)2. Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. However, the molecular mechanism by which FBPA/P achieves its unusual bifunctionality remains unknown. Here we report the crystal structure of FBPA/P at 1.5-Å resolution in the aldolase form, where a critical lysine residue forms a Schiff base with DHAP. A structural comparison of the aldolase form with a previously determined phosphatase form3 revealed a dramatic conformational change in the active site, demonstrating that FBPA/P metamorphoses its active-site architecture to exhibit dual activities. Thus, our findings expand the conventional concept that one enzyme catalyses one biochemical reaction.

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Figure 1: Active sites of ST0318 in the two forms.
Figure 2: Catalytic components of FBPA.
Figure 3: Proposed mechanism of the FBPA and FBPase reactions catalysed by ST0318.

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Protein Data Bank

Data deposits

The atomic coordinates and structure factors are deposited in Protein Data Bank under accession number 3R1M

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Acknowledgements

We thank O. Nureki for discussions, and the staff of the Photon Factory for the X-ray data collection.

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Authors

Contributions

S.F. collected the diffraction data and determined the crystal structure. H.N. prepared the wild-type and mutant proteins and crystallized the wild-type protein. D.H. prepared and crystallized the wild-type protein. D.H. and H.-J.S. measured the enzyme activity. S.F., H.N. and T.W. conceived the project and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Takayoshi Wakagi.

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The authors declare no competing financial interests.

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The file contains Supplementary Tables 1-2, Supplementary Figures 1-8 with legends and Supplementary References. (PDF 4063 kb)

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Fushinobu, S., Nishimasu, H., Hattori, D. et al. Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase. Nature 478, 538–541 (2011). https://doi.org/10.1038/nature10457

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