D'Ambrosio Katia, Pedone Emilia, Langella Emma, De Simone Giuseppina, Rossi Mosè, Pedone Carlo, Bartolucci Simonetta
Istituto di Biostrutture e Bioimmagini, C.N.R., Napoli, Italy.
J Mol Biol. 2006 Sep 29;362(4):743-52. doi: 10.1016/j.jmb.2006.07.038. Epub 2006 Jul 28.
The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.
半胱氨酸残基之间二硫键的形成是蛋白质折叠过程中的限速步骤。为了控制这一氧化过程，不同的生物体发展出了不同的系统。在细菌中，二硫键的形成由Dsb蛋白家族协助；在真核生物中，二硫键的形成和重排由PDI催化。在嗜热生物中，最近一个新的胞质蛋白二硫氧化还原酶家族被认为在二硫键形成中可能起关键作用，该家族成员的分子量约为26 kDa，其特征是具有两个硫氧还蛋白折叠结构，每个折叠结构都包含一个CXXC活性位点基序。在此，我们报道了从嗜热栖热菌K1中分离出的该家族新成员ApPDO的功能和结构特征。功能研究表明，ApPDO可以催化二硫键的还原、氧化和异构化。结构研究表明，该蛋白具有两个CXXC活性位点，其几何参数相当相似，具有典型的稳定构象。最后，对半胱氨酸pK(a)值的理论计算表明，这两个活性位点具有相似的功能特性，且每个活性位点都能赋予酶活性。我们的结果证明了蛋白二硫氧化还原酶家族成员与真核酶PDI之间的功能相似性。然而，由于这两种生物系统不同的三维特征强烈表明其作用机制存在显著差异，因此需要进一步的实验研究来弄清楚不同的三维结构如何导致功能行为相似的系统。
