Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop
Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop
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DOI：
10.1007/s00253-011-3791-4
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发表时间：
2012-08-01
影响因子：
5
通讯作者：
Guo, Rey-Ting
中科院分区：
文献类型：
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作者：
Cheng, Ya-Shan;Ko, Tzu-Ping;Guo, Rey-Ting
来自海栖热袍菌的纤维素酶12 A（TmCel 12 A）是一种超热稳定的β-1，4-内切葡聚糖酶。我们最近确定了TmCel 12 A及其与寡糖的复合物的晶体结构。在这里，通过使用定点诱变，Arg 60和Tyr 61在TmCel 12 A的独特表面环中所起的作用进行了研究。结果与先前观察到的这两个残基和底物之间的氢键和堆积相互作用是一致的。有趣的是，与野生型酶和其他突变体相比，突变体Y 61 G具有最高的活性。它还显示出比野生型更宽的工作温度范围，沿着保持了超热稳定性。Y 61 G的k（cat）和K（m）值均高于野生型。结合Y 61 G-底物复合物的晶体结构，动力学数据表明，更高的内切葡聚糖酶活性可能是由于在还原端裂解的糖部分容易解离。其他晶体学分析表明，Tyr 61位点的插入和缺失突变不会影响整体蛋白质结构，但局部扰动可能会降低底物结合强度。TmCel 12 A的催化效率可能是底物结合和产物释放之间的微妙平衡。Y 61 G单点突变提高酶活力的研究为工程酶的工业化应用提供了一个很好的范例。
Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable beta-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k (cat) and K (m) values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.