Athauda Senarath B P, Matsumoto Koji, Rajapakshe Sanath, Kuribayashi Masayuki, Kojima Masaki, Kubomura-Yoshida Nobuko, Iwamatsu Akihiro, Shibata Chiaki, Inoue Hideshi, Takahashi Kenji
Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Biochem J. 2004 Jul 1;381(Pt 1):295-306. doi: 10.1042/BJ20031575.
Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.
已知食肉植物会分泌酸性蛋白酶来消化猎物（主要是昆虫）以摄取氮。在本研究中，我们首次从马来王猪笼草（当地称为巴杜拉的一种猪笼草）的捕虫笼液中纯化出两种酸性蛋白酶（猪笼草蛋白酶I和II）并使其达到同质，同时研究了它们的酶学和结构特征。两种酶在pH约为2.6时对酸变性血红蛋白的活性最佳；猪笼草蛋白酶I对氧化胰岛素B链的特异性似乎相似，但比其他天冬氨酸蛋白酶（AP）的特异性略宽。然而，在酶学特性中，最值得注意的是它们不同寻常的稳定性：两种酶在50摄氏度及以下都非常稳定，特别是猪笼草蛋白酶I在pH值从3到10的广泛范围内超过30天都极其稳定。这表明这些酶在进化上适应了它们的特定栖息地。我们还从细叶猪笼草的捕虫笼组织中克隆了猪笼草蛋白酶I和II前体的cDNA，并推导了它们的完整氨基酸序列（分别为437和438个残基）。尽管相应的成熟酶（各359个残基）与普通胃蛋白酶型AP同源，但两种酶都含有高含量的半胱氨酸残基（每个分子12个残基），计算机建模表明这些残基可形成六个独特的二硫键，并且这些二硫键被认为有助于猪笼草蛋白酶的显著稳定性。此外，猪笼草蛋白酶与普通AP（包括植物液泡AP）的氨基酸序列同一性非常低（约20%），系统发育比较表明猪笼草蛋白酶与它们的关系较远，形成了一个具有高含量半胱氨酸残基和特征性插入序列的新型AP亚家族，该插入序列名为“猪笼草蛋白酶型AP特异性插入序列”，包括在基因/蛋白质数据库中出现的大量新的直系同源植物AP。
