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首页> 外文期刊>Applied Microbiology >Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability
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Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

机译：钙位点的极性改变诱导疏水相互作用网络并增强Cel9A内切葡聚糖酶的热稳定性

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摘要

Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca~(2+)-free and Ca~(2+)-bounded conformations of the calcium site, we found that Ca~(2+) induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca~(2+)), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca~(2+) added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca~(2+) in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature.
机译：结构性钙位点通过稳定天然折叠和改变局部构象来控制蛋白质的热稳定性和活性。酸热脂环酸杆菌在热酸性条件下存活，并产生内切葡聚糖酶Cel9A（AaCel9A），该酶在催化裂隙附近含有一个钙结合位点（Ser465至Val470）。通过叠加钙位点的无Ca〜（2+）和Ca〜（2+）结合的构象，我们发现Ca〜（2+）通过驱动构象性诱导钙位点及其附近区域之间的疏水相互作用。更改。通过用疏水残基取代周围的极性残基以影响酶的热稳定性和活性，可以进一步增强高B因子区域的疏水相互作用。因此，AaCel9A的钙结合残基Asp468（其侧链直接连接Ca〜（2 +）），Asp469和Asp471分别被丙氨酸和缬氨酸取代。与添加了0 mM或10 mM Ca〜（2+）的野生型相比，突变体D468A和D468V表现出增强的活性，而Asp469或Asp471取代导致活性降低。 D468A的晶体结构表明，突变D468A触发了与野生型Ca〜（2+）诱导的构象变化相似的构象变化，并在钙位点和邻近的疏水区（Ala113至Ala117）之间形成了疏水相互作用网络。突变温度D468V和D468A在熔解温度下分别增加了4.5°C和5.9°C，在75°C下的酶半衰期增加了约13倍。 AaCel9A与其他GH9家族内切葡聚糖酶之间的结构比较表明，对应于AaCel9A钙位点的区域的稳定性在高温下GH9内切葡聚糖酶催化中起着重要作用。

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