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Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states

Nature Structural Biology volume 7, pages 147–153 (2000)Cite this article

Abstract

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 Å crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as ∼30°C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.

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Figure 1: a, Ribbon diagram and b, folding topology of TS-Phy.
Figure 3: High-affinity calcium sites.
Figure 2: Stereo view of Cα trace of TS-Phy structure.
Figure 4: Scatchard plot derived from the duplicate measurements of Ca2+ binding to TS-Phy by a modified equilibrium dialysis method24.
Figure 5: Low-affinity calcium sites.
Figure 6: The dependence of thermostability of TS-Phy on calcium ions as assessed by differential scanning calorimetry.

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Acknowledgements

We would like to thank N. Sakabe for kind help in Synchrotron X-ray data collection and processing, and we gratefully acknowledge the use of the X-ray Facility at Pohang Light Source (PLS) and of the BL6B beamline at Photon Factory in Japan for the X-ray data, and PLS EXAFS beamline 3C1 for the XAS experiment. We also thank S.-S. Yoo for the AAS analysis. This study was supported by the G7 project from Korean Ministry of Science and Technology, and in part by the Sakabe Project of TARA and by the Brain Korea 21 Program of Ministry of Education.

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Authors and Affiliations

  1. Department of Life Science and School of Environmental Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, South Korea

    Nam-Chul Ha, Sejeong Shin, Hyun-Ju Kim, Kwan Yong Choi & Byung-Ha Oh

  2. Microbial enzyme RU, Korea Research Institute of Bioscience & Biotechnology, Taejon, 305-600, P.O. Box 115, Yusong, South Korea

    Byung-Chul Oh, Tae-Kwang Oh & Young-Ok Kim

  3. Center for TARA, University of Tsukuba, Tsukuba, 305, Japan

    Byung-Ha Oh

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Ha, NC., Oh, BC., Shin, S. et al. Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states. Nat Struct Mol Biol 7, 147–153 (2000). https://doi.org/10.1038/72421

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