Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants.
Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants.
复制标题
猪葡萄球菌脂肪酶中钙结合位点的鉴定：钙独立变体的产生。
作者：
J. Simons;M. D. van Kampen;I. Ubarretxena;R. Cox;C. M. Alves dos Santos;M. Egmond;H. M. Verheij
在这项研究中，我们发现猪葡萄球菌脂肪酶中存在钙的高亲和力结合位点。通过等温滴定量热法，我们表明该酶每个酶分子结合一个钙，解离常数为 55 microM。与钙离子存在下的活性相比，脱辅基酶的残余活性从 10 ℃ 时的 65% 到 40 ℃ 时几乎为零。基于与其他葡萄球菌脂肪酶以及来自热链芽孢杆菌和来自荚绿假单胞菌的脂肪酶的一级序列比对并结合定点诱变，天冬氨酸354和357可以被鉴定为钙配体。 D357E 变体的动力学测量表明，用谷氨酸替代 Asp357 可使对钙离子的亲和力降低 30 倍。在位置357处引入赖氨酸、天冬酰胺或丙氨酸以及在位置354处引入赖氨酸或天冬酰胺导致不依赖于钙的变体。等温滴定量热法证实了钙结合的损失。尽管 D357K、D357N 和 D357A 变体不结合钙，但在室温下，它们在钙存在下几乎与野生型脂肪酶一样活跃，但在高温下，这些不依赖于钙的脂肪酶显示出活性降低。在整个温度范围内，在钙存在下，D354K和D354N变体的活性显着低于野生型酶，并且与野生型脱辅基酶的活性相当。我们的结果表明，钙的结合对于葡萄球菌脂肪酶（可能还有其他脂肪酶）的结构稳定很重要，并且可以在与另一种脂肪酶的有限结构同源性的基础上设计不依赖于钙的变体。
In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.