Han X Y, Galloway D R
Department of Microbiology, Ohio State University, Columbus 43210-1292.
J Biol Chem. 1995 Jan 13;270(2):679-84. doi: 10.1074/jbc.270.2.679.
Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of bacterial ADP-ribosylating toxins which use NAD+ as the ADP-ribose donor. By analogy to diphtheria and pertussis toxins, the His440 residue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His440 residue was explored through site-directed mutagenesis which resulted in the production of ETA proteins containing Ala, Asn, and Phe substitutions at the 440 position. The His440-substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed severely reduced ADP-ribosylation activity (> 1000-fold). However, NAD glycohydrolase activity remained intact and in the case of ETAH440N actually increased 10-fold. NAD+ binding is not affected by substitutions at the 440 site as indicated by similar Km values for the ETA variants tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensitive monoclonal antibody as well as sensitivity to proteinase digestion. In view of the location of His440 residue within or close to the proposed NAD(+)-binding site, these results suggest that His440 may be a catalytic residue involved in the transfer of the ADP-ribose moiety to the EF-2 substrate.
铜绿假单胞菌外毒素A（ETA）是细菌ADP核糖基化毒素家族的成员之一，该家族毒素以NAD⁺作为ADP核糖供体。与白喉毒素和百日咳毒素类似，ETA的His440残基被认为是毒素活性位点内的关键残基之一。在本研究中，通过定点诱变探索了His440残基的作用，定点诱变产生了在440位含有丙氨酸、天冬酰胺和苯丙氨酸替代物的ETA蛋白。对His440替代的ETA蛋白进行了纯化和分析。440位点的所有替代物均显示出严重降低的ADP核糖基化活性（>1000倍）。然而，NAD糖水解酶活性保持完整，在ETA-H440N的情况下实际上增加了10倍。如所测试的ETA变体的类似Km值所示，440位点的替代物不影响NAD⁺结合。通过用构象敏感单克隆抗体分析以及对蛋白酶消化的敏感性评估，突变毒素的构象完整性似乎基本未受影响。鉴于His440残基在拟议的NAD⁺结合位点内或附近的位置，这些结果表明His440可能是参与将ADP核糖部分转移至EF-2底物的催化残基。
