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

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 prodomain 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 S 2 and for positively charged residues in S 1 . A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S 2 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.

Long-read sequencing uncovers the adaptive topography of a carnivorous plant genome

“…Several other significantly enriched GOs are associated with this gene family. Cysteine proteases have been identified as major functional components of Venus flytrap (Dionaea muscipula) digestive fluid (72), reported in three D. muscipula transcriptomes (70,73,74), and structurally annotated for both Cape sundew (Drosera capensis) draft genome sequences (75,76) and D. muscipula (77). We found tandem clusters of homologous proteaseencoding genes in the U. gibba genome that had demonstrably undergone tandem duplication both before and after the most recent WGD event in U. gibba's evolutionary history (Fig.…”

Novel proteases from the genome of the carnivorous plant Drosera capensis: Structural prediction and comparative analysis

“…Comparison between our predicted structure (calculated on September 15, 2015), and the crystal structure of Dionain 1 (submitted to the PDB on December 9, 2015), provides a serendipitous opportunity to validate our modeling approach. Because the Dionain 1 structure was submitted to the PDB after the calculation of our predicted structure, it was not in the training set available to Rosetta.…”

“… (a) The predicted Rosetta structure of Dionain 1 (gray) overlaid with the subsequently solved crystal structure (PDBID 1EF7, green) . ( b ) Structural comparison among the predicted structure of Aspain (orange), the structure of Ricinus communis CysEP (CYSEP_RICCO) used by Rosetta as the parent structure of Aspain (PDBID 1S4V, dark blue), and Cathepsin X (PDBID 1EF7, light blue) .…”

The role of electrical and jasmonate signalling in the recognition of captured prey in the carnivorous sundew plant Drosera capensis

“…Following the nomenclature of Takahashi et al (2012), we have identified at least three cysteine proteases called droserains, and one aspartic protease called droserasin was also identified. The digestive fluid of the Venus flytrap contains four cysteine proteases called dionains (recently crystalized by Risør et al, 2016) and two aspartic proteases called dionaeasins (Schulze et al, 2012). In the pitcher plants of the genus Nepenthes, five aspartic proteases called nepenthesins and prolyl-endopeptidase neprosins have been identified (Athauda et al, 2004;Lee et al, 2016), but the presence of a cysteine protease is dubious (Stephenson & Hogan, 2006) because it has never been identified directly in the digestive fluid (Hatano & Hamada, 2008Lee et al, 2016;Rottloff et al, 2016).…”