title: Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics.
abstract: Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and nepenthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50 degrees C both enzymes were remarkably stable; especially Nep 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 Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps 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 Nep-type AP (NAP)-specific insertion', including a large number of novel, orthologous plant APs emerging in the gene/protein databases.
authors.affiliation: Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. kenjitak@ls.toyaku.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Takahashi
authors.forename: Senarath B P
authors.initials: SB
authors.lastname: Athauda
authors.forename: Koji
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Sanath
authors.initials: S
authors.lastname: Rajapakshe
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kuribayashi
authors.forename: Masaki
authors.initials: M
authors.lastname: Kojima
authors.forename: Nobuko
authors.initials: N
authors.lastname: Kubomura-Yoshida
authors.forename: Akihiro
authors.initials: A
authors.lastname: Iwamatsu
authors.forename: Chiaki
authors.initials: C
authors.lastname: Shibata
authors.forename: Hideshi
authors.initials: H
authors.lastname: Inoue
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D019684:Magnoliopsida / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D016282:Aspartic Acid Endopeptidases