Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations
Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations
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DOI：
10.1021/jp200330z
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发表时间：
2011-06-23
影响因子：
3.3
通讯作者：
Polikarpov, Igor
中科院分区：
文献类型：
--
作者：
Bleicher, Lucas;Prates, Erica T.;Polikarpov, Igor
糖基水解酶是能够破坏多糖的糖苷键的酶，并且具有相当大的工业和生物技术应用。在后来的应用的驱动下，经常希望糖基水解酶在极端环境条件下，如高温和极端pH下显示稳定性和活性。在这里，我们提出了X射线结构的超嗜热laminarinase从Rhodothermus marinus（RmLamR）确定在1.95埃的分辨率和分子动力学模拟研究，旨在了解的分子基础，这类酶的热稳定性。与大多数热稳定蛋白一样，RmLamR含有相对大量的盐桥，这些盐桥在结构上不是随机分布的。相反，它们形成簇，使催化结构域的β-片层相互连接。然而，并非所有的盐桥都有利于蛋白质的热稳定性：渗透到酶的疏水核心的电荷-电荷相互作用的存在实际上有助于通过促进水渗透到疏水空腔中来破坏结构，如在嗜温酶的情况下所见。此外，我们证明了侧链的流动性在每一类酶中受到不同的扰动。中温海带酶催化裂周围的环残基侧链在高温下获得移动性并阻碍活性位点。相比之下，嗜热海带酶保留其活性位点的灵活性，活性位点的裂缝仍然可以识别多糖底物，即使在高温下。本研究结果提供了盐桥和活性位点的灵活性对蛋白质热稳定性所起的作用的结构的见解，并可能是相关的其他类别的蛋白质，特别是糖基水解酶。
Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 angstrom resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis, for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting beta-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.