Oda K, Takahashi T, Tokuda Y, Shibano Y, Takahashi S
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
J Biol Chem. 1994 Oct 21;269(42):26518-24.
A unique carboxyl proteinase (EC 3.4.23.33), insensitive to the classical inhibitor isovaleryl pepstatin and isolated from Pseudomonas sp. 101 (PCP), is the first example of a prokaryotic enzyme of this class. The gene coding for PCP was cloned, sequenced, and expressed in Escherichia coli. The gene consists of 1,761 base pairs encoding a protein of 587 amino acid residues. The NH2-terminal 215-amino acid preprosequence flanks the 372-amino acid mature protein, which is identical with the primary structure of an authentic PCP determined by chemical methods. E. coli carrying a plasmid containing the cloned wild-type PCP gene produced a 62-kDa protein. This molecule was processed and secreted into the periplasm as a 43-kDa protein, which converted to mature PCP under acidic conditions. This autocatalytic conversion was completely blocked by tyrostatin, a PCP-specific peptidic inhibitor from Kitasatosporia sp. 55. The purified recombinant PCP has the same characteristics as authentic PCP. When several preprosequence deletion mutants were expressed in E. coli, mutant proteins were accumulated as insoluble forms with no proteinase activities. These results suggest that the prepropeptide of PCP plays an essential role in the formation of functional PCP.
一种独特的羧基蛋白酶（EC 3.4.23.33），对经典抑制剂异戊酰胃蛋白酶抑制素不敏感，从假单胞菌属101菌株（PCP）中分离得到，是此类原核酶的首个实例。编码PCP的基因被克隆、测序并在大肠杆菌中表达。该基因由1761个碱基对组成，编码一个含有587个氨基酸残基的蛋白质。氨基末端的215个氨基酸的前原序列位于372个氨基酸的成熟蛋白两侧，该成熟蛋白与通过化学方法确定的天然PCP的一级结构相同。携带含有克隆的野生型PCP基因的质粒的大肠杆菌产生了一种62 kDa的蛋白质。该分子经过加工后以43 kDa的蛋白质形式分泌到周质中，并在酸性条件下转化为成熟的PCP。这种自催化转化被酪蛋白抑菌素完全阻断，酪蛋白抑菌素是一种来自北里孢菌属55菌株的PCP特异性肽类抑制剂。纯化的重组PCP具有与天然PCP相同的特性。当几种前原序列缺失突变体在大肠杆菌中表达时，突变蛋白以不溶性形式积累，且无蛋白酶活性。这些结果表明，PCP的前原肽在功能性PCP的形成中起重要作用。
