利用定点诱变技术探究产气荚膜梭菌α毒素的结构-功能关系。
Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens.
作者信息
Guillouard I, Garnier T, Cole S T
机构信息
Unité de Génétique Moléculaire Bactaŕienne, Institut Pasteur, Paris, France.
出版信息
Infect Immun. 1996 Jul;64(7):2440-4. doi: 10.1128/iai.64.7.2440-2444.1996.
Abstract
The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available. This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced. Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.
摘要
产气荚膜梭菌α毒素的氨基末端结构域与蜡样芽孢杆菌的完整磷脂酶C（PC-PLC）高度同源，蜡样芽孢杆菌的完整磷脂酶C已有高分辨率晶体结构。该结构信息被用作定点诱变策略的基础，在该策略中，参与锌结合、与底物相互作用或催化的α毒素关键氨基酸残基被替换。对相应毒素变体的生化研究表明，可能存在一个具有卵磷脂酶、鞘磷脂酶和溶血活性的单一活性位点。通过使用一种高度纯化的变体，其中第56位的催化天冬氨酸残基被天冬酰胺取代，结果表明磷脂酶活性对于体内致死性和体外介导血小板聚集至关重要。
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本文引用的文献
1
The biochemistry of bacterial toxins: The lecithinase activity of Cl. welchii toxins.
Biochem J. 1941 Sep;35(8-9):884-902. doi: 10.1042/bj0350884.
2
Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.
J Bacteriol. 1993 Jul;175(14):4298-306. doi: 10.1128/jb.175.14.4298-4306.1993.
3
Purification, crystallization and preliminary X-ray diffraction studies of alpha-toxin of Clostridium perfringens.
J Mol Biol. 1994 Dec 16;244(5):648-50. doi: 10.1006/jmbi.1994.1758.
4
Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis.
Infect Immun. 1994 Nov;62(11):5032-9. doi: 10.1128/iai.62.11.5032-5039.1994.
5
Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin.
J Bacteriol. 1995 Mar;177(5):1179-85. doi: 10.1128/jb.177.5.1179-1185.1995.
6
Effect of some divalent metal cations on phospholipase C from Bacillus cereus.
Acta Chem Scand B. 1981;35(1):39-44. doi: 10.3891/acta.chem.scand.35b-0039.
7
Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme.
Arch Biochem Biophys. 1984 Jun;231(2):400-10. doi: 10.1016/0003-9861(84)90403-x.
8
Rapid and efficient site-specific mutagenesis without phenotypic selection.
Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92. doi: 10.1073/pnas.82.2.488.
9
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.
Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8. doi: 10.1073/pnas.82.4.1074.
10
Lethal effects and cardiovascular effects of purified alpha- and theta-toxins from Clostridium perfringens.
J Infect Dis. 1988 Feb;157(2):272-9. doi: 10.1093/infdis/157.2.272.
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