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首页> 外文期刊>Biochemistry >Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca2+-Driven Catalytic Cycle
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Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca2+-Driven Catalytic Cycle

机译：来自Saccharomonospora Viridis AHK190在底物结合状态下的宠物降解的皮酶样酶的结构动力学阐明了Ca2 + -drive催化循环

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摘要

A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca2+ binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca2+ binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca2+ binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca2+ can greatly accelerate.
机译：已显示来自Saccharomonospora Viridis AHK190（Cut190）的Cutinase型聚酯酶，从而降解聚对苯二甲酸乙二醇酯的内部块。 Cut190的独特特征是其功能和稳定性由CA2 +绑定调节。我们之前的Cut190S226P的晶体结构分析表明，一个Ca2 +与酶结合，其在几个环区域中诱导大构象变化以稳定开放构象[Miyakawa，T.等人。 （2015）应用。微生物。 Biotechnol。 99,4297]。在该研究中，为了分析切割190的基板识别机制，我们确定了与钙离子和/或衬底复合物的切割190突变体，切割190 * S176a的无活性形式的晶体结构。我们发现三个钙离子与Cut190 * S176a结合，通过使用天然质谱实验和3D参考交互站点计算通过分析来支持。具有两种底物，单乙基琥珀酸盐和单乙基（接合和开放形式）的复合结构可能对应于反应前和后后状态，因为酯键接近活性位点并从活性位点向外指向，分别为两种复合物。 CA2 +装订会使口袋打开，使基板能够更容易地进入口袋。分子动力学模拟表明，在接合形式的后反应状态中的实验观察到的形式之间可能存在，这表明所述基板将在接合形式被切割，然后需要酶来改变到打开形式释放产品，一个CA2 +可以大大加速的过程。

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  • 来源
    《Biochemistry》 |2018年第36期|共12页
  • 作者

    Numoto Nobutaka; Kamiya Narutoshi; Bekker Gert-Jan; Yamagami Yuri; Inaba Satomi; Ishii Kentaro; Uchiyama Susumu; Kawai Fusako; Ito Nobutoshi; Oda Masayuki;

  • 作者单位

    Tokyo Med &

    Dent Univ Med Res Inst Bunkyo Ku 1-5-45 Yushima Tokyo 1138510 Japan;

    Osaka Univ Inst Prot Res 3-2 Yamadaoka Suita Osaka 5650871 Japan;

    Osaka Univ Inst Prot Res 3-2 Yamadaoka Suita Osaka 5650871 Japan;

    Kyoto Prefectural Univ Grad Sch Life &

    Environm Sci Sakyo Ku 1-5 Hangi Cho Kyoto Kyoto 6068522 Japan;

    Kyoto Prefectural Univ Grad Sch Life &

    Environm Sci Sakyo Ku 1-5 Hangi Cho Kyoto Kyoto 6068522 Japan;

    Natl Inst Nat Sci Exploratory Res Ctr Life &

    Living Syst 5-1 Higashiyama Okazaki Aichi 4448787 Japan;

    Natl Inst Nat Sci Exploratory Res Ctr Life &

    Living Syst 5-1 Higashiyama Okazaki Aichi 4448787 Japan;

    Kyoto Inst Technol Ctr Fiber &

    Text Sci Sakyo Ku Kyoto Kyoto 6068585 Japan;

    Tokyo Med &

    Dent Univ Med Res Inst Bunkyo Ku 1-5-45 Yushima Tokyo 1138510 Japan;

    Kyoto Prefectural Univ Grad Sch Life &

    Environm Sci Sakyo Ku 1-5 Hangi Cho Kyoto Kyoto 6068522 Japan;

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  • 正文语种 eng
  • 中图分类 生物化学;
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