Nepenthesin, a Unique Member of a Novel Subfamily of Aspartic Proteinases: Enzymatic and Structural Characteristics

Kenji      Takahashi; Senarath   B. P.   Athauda; Koji      Matsumoto; Sanath      Rajapakshe; Masayuki      Kuribayashi; Masaki      Kojima; Nobuko      Kubomura-Yoshida; Akihiro      Iwamatsu; Chiaki      Shibata; Hideshi      Inoue

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°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.

Keywords: aspartic proteinase, carnivorous plant, characterization, Nepenthes, nepenthesin, plant proteinase

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