Abstract
Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.
Highlights
Including the passive pitfall traps of tropical pitcher plants (Nepenthes), the adhesive flypaper traps of sundews (Drosera), and the active snap traps of Venus flytraps
Dionains display Ͻ70% sequence similarity to other cysteine proteases characterized to date. They are ascribed to the papain-like C1 family of the MEROPS database according to the overall homology and conservation of specific sequence motifs uniquely related to autocatalytic maturation from inactive zymogenic pro-forms to active proteases [14]
De Novo Sequencing of Full-length Pre-pro-dionain-1—The most abundant protein in the digestive fluid of the Venus flytrap, based on SDS-PAGE and Coomassie Brilliant Blue staining, is a ϳ45-kDa cysteine protease named dionain-1, reflecting its origin (Dionaea) and homology with papain [41]
Summary
Materials—Venus flytrap plants were purchased from the Lammehave Nursery (Ringe, Denmark) and grown in a walk-in plant growth chamber under a 12-h/12-h light/dark cycle at 26 °C. The LA PCR kit was purchased from Clontech, and the FastDigest restriction enzymes and T4 ligase were obtained from Fermentas. HiTrap columns, Superdex 75 10/30, and the Ettan CAFTM MALDI sequencing kit were purchased from GE Healthcare. Z-FR-AMC2 was obtained from Bachem, and Z-LR-AMC, BocLKR, and Boc-GKR were purchased from the Peptide Institute Inc. All other chemicals were obtained from Sigma-Aldrich unless otherwise specified. De Novo Sequencing of Dionain-1 Peptides—Digestive fluid from mechanostimulated Venus flytrap leaves was analyzed by SDS-PAGE using a 5–15% gradient gel. The suspected major band containing dionain-1 was excised for in-gel trypsin digestion or analyzed using automated Edman degradation (Procise 494-HT protein sequencer) after electrophoretic blotting to an Immobilon membrane. The trypsin digestion was performed overnight at 37 °C and followed by acidification with 0.1% trifluoroacetic acid (TFA), C18 reverse-phase micropurification,
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