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 °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.
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Research Article|
June 22 2004
Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases
Senarath B. P. ATHAUDA;
Senarath B. P. ATHAUDA
*Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
†Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Peradeniya and Institute of Fundamental Studies, Kandy, Sri Lanka
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Koji MATSUMOTO;
Koji MATSUMOTO
*Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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Sanath RAJAPAKSHE;
Sanath RAJAPAKSHE
†Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Peradeniya and Institute of Fundamental Studies, Kandy, Sri Lanka
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Masayuki KURIBAYASHI;
Masayuki KURIBAYASHI
*Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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Masaki KOJIMA;
Masaki KOJIMA
*Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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Nobuko KUBOMURA-YOSHIDA;
Nobuko KUBOMURA-YOSHIDA
‡Central Laboratories for Key Technology, Kirin Brewery Co., Yokohama, Kanagawa 236-0004, Japan
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Akihiro IWAMATSU;
Akihiro IWAMATSU
‡Central Laboratories for Key Technology, Kirin Brewery Co., Yokohama, Kanagawa 236-0004, Japan
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Chiaki SHIBATA;
Chiaki SHIBATA
§Department of Biology, Nippon Dental University, Chiyoda-ku, Tokyo 102-8159, Japan
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Hideshi INOUE;
Hideshi INOUE
*Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
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Kenji TAKAHASHI
Kenji TAKAHASHI
1
*Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
October 15 2003
Revision Received:
March 22 2004
Accepted:
March 22 2004
Accepted Manuscript online:
March 23 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 381 (1): 295–306.
Article history
Received:
October 15 2003
Revision Received:
March 22 2004
Accepted:
March 22 2004
Accepted Manuscript online:
March 23 2004
Connected Content
A correction has been published:
Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases
Citation
Senarath B. P. ATHAUDA, Koji MATSUMOTO, Sanath RAJAPAKSHE, Masayuki KURIBAYASHI, Masaki KOJIMA, Nobuko KUBOMURA-YOSHIDA, Akihiro IWAMATSU, Chiaki SHIBATA, Hideshi INOUE, Kenji TAKAHASHI; Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases. Biochem J 1 July 2004; 381 (1): 295–306. doi: https://doi.org/10.1042/BJ20031575
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