title: Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases.
abstract: 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.
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.
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
authors.forename: Kenji
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D019684:Magnoliopsida / Q000166:cytology / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009646:Norleucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010084:Oxidation-Reduction; D010436:Pepstatins / Q000494:pharmacology; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D018744:DNA, Plant; D007328:Insulin; D010436:Pepstatins; D010940:Plant Proteins; C000909:N-diazoacetylnorleucine methyl ester; D009646:Norleucine; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin