Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.
Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.
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DOI：
10.1021/bi401561p
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发表时间：
2014-03
期刊：
影响因子：
2.9
通讯作者：
Sintawee Sulaiman;D. You;E. Kanaya;Y. Koga;S. Kanaya
中科院分区：
文献类型：
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作者：
Sintawee Sulaiman;D. You;E. Kanaya;Y. Koga;S. Kanaya
在1.5 nm分辨率下测定了具有聚对苯二甲酸乙二醇酯（PET）降解活性的宏基因组衍生的LC-角质酶的晶体结构。结构非常类似于Thermobifida alba角质酶。Ser 165、Asp 210和His 242形成催化三联体。采用圆二色谱法研究了pH 8.0时LC-角质酶的热变性和盐酸胍诱导的去折叠。在30 °C下，热变性曲线的转变的中点T1/2和GdnHCl诱导的解折叠曲线的中点Cm分别为86.2 °C和4.02 M。在30 °C下，在没有盐酸钆的情况下，去折叠的自由能变化ΔG（H2O）为41.8 kJ mol（-1）。LC-角质酶在GdnHCl中的解折叠速度非常慢，50 °C时的解折叠速率ku（H2O）为3.28 × 10（-6）s（-1）。这些结果表明LC-角质酶是动力学稳健的蛋白质。然而，LC-角质酶对对硝基苯基丁酸酯的活性的最适温度（50 °C）显著低于T1/2值。在1%聚乙二醇（PEG）1000的存在下，它增加了10 °C。当PET用作基材时，它也增加了至少20 °C。这些结果表明，活性位点被保护从热诱导的局部构象变化的PEG或PET的结合。LC-角质酶在Cys 275和Cys 292之间含有一个二硫键。为了研究这种二硫键是否有助于LC-角质酶的热力学和动力学稳定性，构建了没有这种二硫键的C275/292 A-角质酶。热变性研究和平衡和动力学研究的钆盐酸诱导的C275/292 A-角质酶的去折叠表明，这种二硫键不仅有助于热力学稳定性，但也LC-角质酶的动力学稳定性。
The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.