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Structural basis for the Ca2+-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190

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Abstract

A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an α/β hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca2+-induced thermostabilization and activation of enzymes have been well explored in α-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca2+ enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190S226P) in the Ca2+-bound and free states at 1.75 and 1.45 Å resolution, respectively. Based on the crystallographic data, a Ca2+ ion was coordinated by four residues within loop regions (the Ca2+ site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca2+ to Cut190S226P induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca2+ not only stabilized a region that is flexible in the Ca2+-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca2+-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190S226P is activated by a conformational change in the active-site sealing residue, F106.

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Acknowledgments

This work was supported by the Platform for Drug Design, Discovery and Development of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT). We would like to thank the scientists and staff at the Photon Factory. The synchrotron radiation experiments were conducted at the beamlines AR-NW12 and BL-17A at the Photon Factory, Tsukuba, Japan (Proposal No. 2013G658).

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Authors and Affiliations

  1. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Takuya Miyakawa, Hiroki Mizushima, Jun Ohtsuka & Masaru Tanokura

  2. Laboratory of Biophysical Chemistry, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan

    Masayuki Oda

  3. Center for Fiber and Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan

    Fusako Kawai

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  1. Takuya Miyakawa
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  2. Hiroki Mizushima
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  3. Jun Ohtsuka
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  4. Masayuki Oda
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  5. Fusako Kawai
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  6. Masaru Tanokura
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Correspondence to Masaru Tanokura.

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Miyakawa, T., Mizushima, H., Ohtsuka, J. et al. Structural basis for the Ca2+-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190. Appl Microbiol Biotechnol 99, 4297–4307 (2015). https://doi.org/10.1007/s00253-014-6272-8

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  • DOI: https://doi.org/10.1007/s00253-014-6272-8

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