Oda K, Ito M, Uchida K, Shibano Y, Fukuhara K, Takahashi S
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology.
J Biochem. 1996 Sep;120(3):564-72. doi: 10.1093/oxfordjournals.jbchem.a021451.
Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, is the second example of the unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to the classical aspartic proteinase inhibitor. The gene coding for XCP was cloned, sequenced, and expressed in Escherichia coli. The XCP gene contains an open reading frame of 2,481 base pairs encoding a protein of 827 amino acid residues with a M(r) of 83,677. The XCP was synthesized as a large precursor consisting of three regions: NH2-terminal prepro (N-Prepro) (237 amino acid residues); mature XCP (398 a.a.residues); and COOH-terminal pro (C-Pro) (192 a.a. residues). The N-Prepro and mature XCP regions had no sequence similarity to any other proteins reported so far, except the carboxyl proteinase from Pseudomonas sp. 101 [Oda, K., Takahashi, T., Tokuda, Y., Shibano, Y., and Takahashi, S. (1994) J. Biol. Chem. 269, 26518-26524]. The C-Pro region showed high similarity to COOH-terminal regions of other microbial proteinase precursors. E. coli carrying a plasmid containing the cloned wild-type XCP gene produced an 84-kDa protein. This protein was processed into a mature, active form under acidic conditions. This process was completely blocked by tyrostatin, an XCP-specific inhibitor from Kitasatosporia sp. 55, indicating an autocatalytic processing. The purified recombinant XCP had the same characteristics as authentic XCP except for the NH2-terminal amino acid sequence. When the mutant XCP gene truncated in the C-Pro region was expressed in E. coli, an expected 64-kDa protein was detected in the cells, and also processed into the 42-kDa active form under the acidic conditions. Thus, the C-Pro region was not essential for the formation of active mature XCP.
从黄单胞菌属菌株T-22中分离得到的黄单胞菌羧基蛋白酶（XCP），是独特的羧基蛋白酶[EC 3.4.23.33]中的第二个例子，这类酶对经典的天冬氨酸蛋白酶抑制剂不敏感。编码XCP的基因被克隆、测序，并在大肠杆菌中表达。XCP基因包含一个2481个碱基对的开放阅读框，编码一个827个氨基酸残基的蛋白质，分子量为83677。XCP以一种由三个区域组成的大前体形式合成：氨基末端前原（N-前原）（237个氨基酸残基）；成熟的XCP（398个氨基酸残基）；以及羧基末端原（C-原）（192个氨基酸残基）。除了来自假单胞菌属菌株101的羧基蛋白酶外，N-前原和成熟XCP区域与迄今报道的任何其他蛋白质均无序列相似性[小田，K.，高桥，T.，德田，Y.，柴野，Y.，和高桥，S.（1994）《生物化学杂志》269，26518 - 26524]。C-原区域与其他微生物蛋白酶前体的羧基末端区域具有高度相似性。携带含有克隆的野生型XCP基因的质粒的大肠杆菌产生了一种84 kDa的蛋白质。这种蛋白质在酸性条件下被加工成成熟的活性形式。这个过程被来自北里孢菌属菌株55的XCP特异性抑制剂酪抑素完全阻断，表明这是一个自催化加工过程。纯化的重组XCP除了氨基末端氨基酸序列外，具有与天然XCP相同的特性。当在C-原区域截短的突变XCP基因在大肠杆菌中表达时，在细胞中检测到一种预期的64 kDa蛋白质，并且在酸性条件下也被加工成42 kDa的活性形式。因此，C-原区域对于活性成熟XCP的形成不是必需的。
