Biochem. J. (2004) 381
(295–306) (Printed in Great Britain)
Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases
Senarath B. P. ATHAUDA*†, Koji MATSUMOTO*, Sanath RAJAPAKSHE†, Masayuki KURIBAYASHI*, Masaki KOJIMA*, Nobuko KUBOMURA-YOSHIDA‡, Akihiro IWAMATSU‡, Chiaki SHIBATA§, Hideshi INOUE* and 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, †Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Peradeniya and Institute of Fundamental Studies, Kandy, Sri Lanka, ‡Central Laboratories for Key Technology, Kirin Brewery Co., Yokohama, Kanagawa 236-0004, Japan, and §Department of Biology, Nippon Dental University, Chiyoda-ku, Tokyo 102-8159, Japan
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.
Key words: aspartic proteinase, carnivorous plant, characterization, Nepenthes, nepenthesin, plant proteinase.
Abbreviations used: AP, aspartic proteinase; CND41, chloroplast nucleoid DNA-binding protein 41; Cya, cysteic acid; DAN, diazoacetyl-D,L-norleucine methyl ester; NAP, nepenthesin-type AP.
1To whom correspondence should be addressed (e-mail kenjitak@ls.toyaku.ac.jp).
The nucleotide sequence data for nepenthesins I and II are available from the DDBJ data bank under the accession numbers AB114914 and AB114915 respectively.
Received 15 October 2003/22 March 2004; accepted 22 March 2004
Published as BJ Immediate Publication 23 March 2004, DOI 10.1042/BJ20031575
The Biochemical Society, London ©2004
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