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.

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

“…In addition to proteins identified in this study (Supplementary Tables 25-28), we obtained for phylogenetic analyses a number of previously published sequences of digestive fluid proteins 8,9,[72][73][74][75][76][77][78] (Supplementary Table 24). Although many protein and transcript sequences for possible digestive enzymes are available (for example, refs 17,79-84 ), we included only genes for which complete coding sequences were available and for which their presence in digestive fluid had been biochemically validated (Supplementary Table 24, last searched 20 January 2016).…”

Stability Profiles of Nepenthesin in Urea and Guanidine Hydrochloride: Comparison with Porcine Pepsin A

“…2,3) On the other hand, when crude fluid of N. alata was incubated at pH 2.9 at 37 C, activity decreased only very slowly on longer incubation. About 60% and 30% of the original activity were retained after 30 d and 65 d of incubation respectively.…”

“…2,3) Although the enzymes share active site motifs, including two catalytic aspartic acid residues and the flap tyrosine residue, with pepsintype aspartic peptidases (MEROPS subfamily A1A), they are different in several other respects. Among these, a higher content of cysteine residues and remarkable stability at relatively high temperatures over a wide range of pH levels are notable.…”

“…Nepenthesin was purified from the pitcher fluid of N. alata essentially as described previously 2) for the enzymes from N. distillatoria, with various modifications. Batchwise treatment with DEAE-cellulose and chromatography on Sephacryl S-200 were omitted.…”

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…The degradation of prey proteins is performed by cysteine endopeptidases (dionains) supported by the action of a serine carboxypeptidase (5). This is in contrast to the digestive fluids of Nepenthes, Cephalotus, and Drosera, which all rely heavily on aspartate proteases (12,13).…”