Structural and functional analysis of pullulan hydrolyzing alpha-amylase and conversion of the substrate specificity

普通嗜热放线菌R-47α-淀粉酶II(TVA II)不仅能降解淀粉，还能降解普鲁兰多糖。无论是α-1，4还是α-1，6糖苷键，这种酶都能水解寡糖麦芽糖单元旁边的糖苷键。以往的研究表明，从202到206的HKYDT序列对底物识别是重要的。在本研究中，这些氨基酸残基被突变为Trp和Ala，并对这些酶的性质进行了动力学检测。研究结果总结如下。(1)所有的色氨酸突变酶几乎失去了全部活性，表明了这些残基的重要性。(2)与野生酶相比，H201A、D205A和T206A的活性没有明显下降。这些残基对活性可能并不重要。(3)突变体H202a对低分子底物的Km值显著降低。(4)突变体K203A对所有底物的Km值均显著增加。(5)Y204A突变体的kcat值在所有底物上均显著降低。考虑到TVA II的三维结构，这些证据可以解释如下。H202和Y204的侧链分别与进入亚位点-2和亚位点-1的糖相互作用。K203的侧链通过与主链形成氢键网络来稳定底物结合部位。为了获得大量用于结晶的蛋白质，我们尝试利用pET系统构建高效表达载体。在含有该质粒的大肠杆菌细胞中可产生大量的蛋白质，但以包涵体形式存在。以前的方法得到的纯酶数量有限，无法对结晶条件进行详细的检查。为了比较TVA II与其他普鲁兰多糖水解酶的结构，我们从嗜热菌Thermus thermophilus HB8中克隆了普鲁兰聚糖酶基因。

Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) hydrolyzes not only starch but also pullulan. This enzyme hydrolyzes glucosidic linkage next to maltose unit of various oligosaccharides whether this is α-1,4 or α-1,6 glucosidic linkage. Previous studies suggested that HKYDT sequence from 202 to 206 is important to the substrate recognition. In this study, those amino acid residues are mutated to Trp and Ala, and the properties of those enzymes are examined kinetically. The results are summarized as follows. (1) All the Trp mutant enzymes lost almost all the activity, suggesting the importance of those residues. (2) The activities of H201A, D205A, and T206A did not so decrease compared to that of wild enzyme. Those residues may not be important to the activity. (3) The Km values of H202A mutant for low molecular weight substrates decreased greatly. (4) The Km values of K203A mutant for all the substrate examined increased remarkably. (5) The kcat values of Y204A mutant drastically decreased for all the substrates. Those evidences can be explained considering the 3D structure of TVA II as follows. The side chain of H202 and Y204 interact with the sugars that enter subsite-2 and subsite-1, respectively. The side chain of K203 stabilizes the substrate binding site by forming hydrogen bond network with the main chain. To obtain large amount of the protein for crystallization, we tried to construct an over-expression vector using pET system. Large amount of protein was produced in Escherichia coli cells harboring the plasmid, but in an inclusion body. The limited amount of the pure enzyme obtained by the previous methods prevented detailed examination of the crystallization condition. To compare the structure of TVA II with other pullulan hydrolyzing enzymes, we cloned a pullulanase gene from a thermophilic bacterium Thermus thermophilus HB8.