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Structural framework of fructosyl transfer in Bacillus subtilis levansucrase

Nature Structural & Molecular Biology volume 10, pages 935–941 (2003)Cite this article

Abstract

Many bacteria and about 40,000 plant species form primary carbohydrate reserves based on fructan; these polymers of β-D-fructofuranose are thought to confer tolerance to drought and frost in plants. Microbial fructan, the β(2,6)-linked levan, is synthesized directly from sucrose by levansucrase, which is able to catalyze both sucrose hydrolysis and levan polymerization. The crystal structure of Bacillus subtilis levansucrase, determined to a resolution of 1.5 Å, shows a rare five-fold β-propeller topology with a deep, negatively charged central pocket. Arg360, a residue essential for polymerase activity, lies in a solvent-exposed site adjacent to the central pocket. Mutagenesis data and the sucrose-bound structure of inactive levansucrase E342A, at a resolution of 2.1 Å, strongly suggest that three conserved acidic side chains in the central pocket are critical for catalysis, and presumably function as nucleophile (Asp86) and general acid (Glu342), or stabilize the transition state (Asp247).

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Figure 1: Sequence of B. subtilis levansucrase and secondary structure assignment.
Figure 2: Electron density of the Ca2+ and sucrose binding sites of B. subtilis levansucrase.
Figure 3: Ribbon diagram of levansucrase and superimposition with Cellvibrio japonicus α-L-arabinanase A43 (Arb43A).
Figure 4: Details of the active site of levansucrase.
Figure 5: Activity assay probing levan synthesis.

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Acknowledgements

This work was supported by funds from the Royal Society and the School of Biosciences. G.M. is a recipient of the Adrian Brown Scholarship. We thank O.C. Mather for help with data collection and staff at ESRF for support at the beamlines. We used a computer cluster funded by the UK Medical Research Council and GlaxoWellcome, and received much valued technical advice from O.S. Smart, A.J. Pemberton and C. Cureton. We are indebted to R. Chambert for valuable discussions, to G. Waksman, J.B. Jackson, C.W. Wharton and S.A. White for comments on the manuscript and to L.M. Machesky and R.H. Insall for sharing their equipment.

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  1. School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK

    Guoyu Meng & Klaus Fütterer

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Correspondence to Klaus Fütterer.

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Meng, G., Fütterer, K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Mol Biol 10, 935–941 (2003). https://doi.org/10.1038/nsb974

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