National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
J Bacteriol. 2010 Nov;192(21):5799-805. doi: 10.1128/JB.00485-10. Epub 2010 Sep 10.
The lethal factor (LF) of Bacillus anthracis is a Zn(2+)-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.
炭疽杆菌的致死因子（LF）是一种依赖 Zn(2+)的金属蛋白酶，在炭疽毒力中发挥重要作用。本研究旨在鉴定 LF 的催化活性所必需的组氨酸残基。通过定点突变分别将 LF 结构域 II、III 和 IV 中的 10 个组氨酸残基突变为丙氨酸残基。测试了这些突变体的细胞毒性，结果表明，组氨酸 669 突变为丙氨酸完全消除了对致死毒素（LT）敏感的 RAW264.7 细胞的毒性。进一步探讨了毒性丧失的原因。该 LF 突变体的锌含量与野生型相同。此外，该 LF 突变体保留了其保护性抗原（PA）结合活性。最后，证明该突变体的催化裂解活性大大降低。因此，我们得出结论，残基 His-669 对 LF 的蛋白水解活性至关重要。
