Kops O, Eckerskorn C, Hottenrott S, Fischer G, Mi H, Tropschug M
Institut für Biochemie und Molekularbiologie der Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
J Biol Chem. 1998 Nov 27;273(48):31971-6. doi: 10.1074/jbc.273.48.31971.
Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes capable of isomerizing a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. Eucaryotic members of the parvulin family have recently been shown to be essential for regulation of mitosis. Here we describe the purification and characterization of Ssp1, an abundant parvulin homolog from Neurospora crassa, which is unique among the known eucaryotic parvulins in containing a polyglutamine stretch between the N-terminal WW domain and the C-terminal PPIase domain. Ssp1 is a site-specific PPIase with respect to the amino acid N-terminal to the proline residue. Peptides with glutamate, phosphoserine, or phosphothreonine in the -1-position proved to be the best substrates. Ssp1 is not only able to isomerize small peptides but is also active in protein folding, as shown with mouse dihydrofolate reductase. Using the substrate specificity of Ssp1, we could identify Glu81-Pro82 as a PPIase-sensitive site in folding of dihydrofolate reductase. These results demonstrate that Ssp1 is a potent mediator of protein folding and that parvulins can serve as tools to elucidate rate-limiting steps in protein folding reactions.
肽基脯氨酰顺反异构酶（PPIases）是能够使Xaa-Pro肽键异构化的酶。已知有三类PPIases：亲环蛋白、FK506结合蛋白和小菌素。人们对PPIases的生理功能了解甚少。最近发现小菌素家族的真核成员对于有丝分裂的调控至关重要。在此，我们描述了粗糙脉孢菌中一种丰富的小菌素同源物Ssp1的纯化和特性，它在已知的真核小菌素中是独特的，在N端WW结构域和C端PPIase结构域之间含有一段聚谷氨酰胺序列。就脯氨酸残基N端的氨基酸而言，Ssp1是一种位点特异性PPIase。在-1位带有谷氨酸、磷酸丝氨酸或磷酸苏氨酸的肽被证明是最佳底物。如小鼠二氢叶酸还原酶所示Ssp1不仅能够使小肽异构化，而且在蛋白质折叠中也具有活性。利用Ssp1的底物特异性，我们能够确定二氢叶酸还原酶折叠过程中Glu81-Pro82是一个PPIase敏感位点。这些结果表明Ssp-1是蛋白质折叠的有效介质，并且小菌素可以作为阐明蛋白质折叠反应限速步骤的工具。
