Key Lab of Microbial Molecular Biology of Hunan Province, College of Life Science, Hunan Normal University, Changsha, 410081, People's Republic of China.
Curr Microbiol. 2011 Mar;62(3):968-73. doi: 10.1007/s00284-010-9791-2. Epub 2010 Nov 17.
Domain III of Bacillus thuringiensis Cry δ-endotoxins are considered to be related to the stability of the structure and avoidance of overdigestion by proteases. In this study, some residues of potential chymotrypsin and trypsin sites in Domain III of B. thuringiensis Cry1Aa were replaced individually with alanine by site-directed mutagenesis, in order to investigate their functional roles. Except F574A, all mutants F536A, R543A, F550A, F565A, R566A, F570A, F576A, F583A, and F590A were highly expressed the 130 kD protoxins at levels comparable to the wild-type tested by SDS-PAGE. In bioassays, F536A, R566A, and F590A increased toxicity against Spodoptera exigua Hüner larve by 20, 40, and 40%, respectively, as compared to the wild-type. F536A and F565A showed an increase of 6 and 10% in toxicity against Heliothis armigera Hubner than the wild-type. Toxicities of some mutants were altered greatly, and the same mutants were shown to have different toxicities against those two insects. Structural analyses showed that mutants R543A, F574A, F576A-affecting insecticidal activity might be relational to structural stability of toxin or decreased affinity for receptor binding. These results indicated that those residues were involved in the larvicidal activity of the Cry1Aa toxin.
苏云金芽孢杆菌 Cry δ-内毒素的结构域 III 被认为与结构的稳定性以及避免被蛋白酶过度消化有关。在这项研究中，通过定点突变技术将苏云金芽孢杆菌 Cry1Aa 结构域 III 中潜在的胰凝乳蛋白酶和胰蛋白酶位点的一些残基分别突变为丙氨酸，以研究它们的功能作用。除 F574A 外，所有突变体 F536A、R543A、F550A、F565A、R566A、F570A、F576A、F583A 和 F590A 都以与野生型相当的水平高度表达了 130 kD 的原毒素，通过 SDS-PAGE 检测。在生物测定中，与野生型相比，F536A、R566A 和 F590A 对甜菜夜蛾幼虫的毒性分别提高了 20%、40%和 40%。F536A 和 F565A 对棉铃虫的毒性比野生型分别提高了 6%和 10%。一些突变体的毒性发生了很大的变化，相同的突变体对这两种昆虫的毒性也不同。结构分析表明，突变体 R543A、F574A、F576A 影响杀虫活性可能与毒素结构稳定性或与受体结合亲和力降低有关。这些结果表明，这些残基参与了 Cry1Aa 毒素的杀虫活性。
