Munier H, Bouhss A, Gilles A M, Krin E, Glaser P, Danchin A, Bârzu O
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Eur J Biochem. 1993 Oct 15;217(2):581-6. doi: 10.1111/j.1432-1033.1993.tb18280.x.
The catalytic domain of Bordetella pertussis adenylate cyclase, a calmodulin-activated enzyme with toxic properties, is a modular construct cleaved by trypsin into two subdomains of 224 (T25) and 175 (T18) amino acids. The calmodulin-binding locus of the bacterial enzyme consists of approximately 70 amino acids and overlaps the C-terminus of T25 and the N-terminus of T18. This region, exposed to the solvent or proteases, also exhibits an unusual high flexibility and allows, as demonstrated in this study, reconstitution in the presence of calmodulin of active species of adenylate cyclase from overlapping inactive fragments of the enzyme. Moreover, several combinations of inactive variants of the bacterial enzyme obtained by site-directed mutagenesis can yield active species. Heterodimers, resulting from a few selected combinations of inactive species of adenylate cyclase, exhibit specific activity similar to that of the native enzyme. Productive complementation from inactive fragments is a unique phenomenon among calmodulin-activated enzymes and represents a new and helpful tool in the understanding of the molecular mechanism of activation of B. pertussis adenylate cyclase upon binding of calmodulin.
百日咳博德特氏菌腺苷酸环化酶的催化结构域是一种具有毒性的钙调蛋白激活酶，它是一种模块化结构，可被胰蛋白酶切割成两个分别含有224个氨基酸（T25）和175个氨基酸（T18）的亚结构域。细菌酶的钙调蛋白结合位点由大约70个氨基酸组成，与T25的C末端和T18的N末端重叠。该区域暴露于溶剂或蛋白酶中时，还表现出异常高的灵活性，并且如本研究所示，在钙调蛋白存在的情况下，可从该酶的重叠无活性片段中重组出有活性的腺苷酸环化酶物种。此外，通过定点诱变获得的细菌酶无活性变体的几种组合可产生有活性的物种。由腺苷酸环化酶无活性物种的一些选定组合产生的异二聚体表现出与天然酶相似的比活性。无活性片段的有效互补是钙调蛋白激活酶中一种独特的现象，是理解钙调蛋白结合后百日咳博德特氏菌腺苷酸环化酶激活分子机制的一种新的有用工具。
