Goh Poh Hong, Illias Rosli Md, Goh Kian Mau
Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
Int J Mol Sci. 2012;13(5):5307-5323. doi: 10.3390/ijms13055307. Epub 2012 Apr 25.
Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
与环糊精葡萄糖基转移酶（CGTase）钙结合位点工程相关的研究有限。在本研究中，对已知具有热稳定性功能的钙结合区域进行了定点诱变。起始基因-蛋白质是CGTase芽孢杆菌属G1的一个变体，该变体先前已有报道，本文中称为“亲本CGTase”。构建了四种CGTase变体（S182G、S182E、N132R和N28R）。在与Ca-I位点和活性位点裂隙相邻的第182位残基处发生突变的两种变体具有增强的热稳定性特征。变体S182G在60℃下的活性半衰期增加到94分钟，而亲本CGTase仅为22分钟。这种改善可能归因于较短α-螺旋的形成以及相应区域甘氨酸对不利空间应变的缓解。对于变体S182E，A/B结构域界面处额外的离子相互作用将半衰期提高到31分钟，但降低了CGTase活性。通过N132R突变在Ca-I位点引入离子相互作用破坏了CGTase的催化活性。相反，在Ca-II位点具有额外离子相互作用的变体N28R显示出环化活性增加。然而，热稳定性未受影响。
