Laboratoire de Métabolites Et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, BP 1177, 3018, Sfax, Tunisia.
J Ind Microbiol Biotechnol. 2013 Sep;40(9):947-53. doi: 10.1007/s10295-013-1300-8. Epub 2013 Jun 15.
Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the K(m) values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward β-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.
来自芽孢杆菌 US149 的麦芽寡糖基淀粉酶（MAUS149）是一种环糊精（CD）降解酶，对 CD 具有很高的偏好性，而对麦芽寡糖的偏好性较低。在本研究中，我们通过定点突变研究了残基 Asp46 在 MAUS149 的特异性和催化特性中的作用。构建并研究了三个突变酶（D46V、D46G 和 D46N）。发现这三种突变酶在热活性、热稳定性和 pH 谱方面与野生型 MAUS149 相似。然而，与野生型相比，突变酶 D46V 和 D46G 对所有底物的动力学参数都发生了巨大改变。事实上，MAUS149/D46G 对所有底物的 K(m) 值都大大增加。然而，与 MAUS149 相比，MAUS149/D46V 对β-CD 的亲和力和催化效率提高了五倍。分子建模表明，残基 D46 与残基 K282 形成盐桥。该键将保持侧链残基 Y45 和 W47 的排列在特定的方向，从而促进进入催化位点并保持其中的底物。因此，任何不带电荷的氨基酸取代都会影响底物结合裂缝中门壁的灵活性，从而导致底物选择性的变化。
