Enzymatic and structural characterization of the major endopeptidase in the Venus flytrap digestion fluid

Michael W Risor, Line R Thomsen, Kristian W Sanggaard, Tania A Nielsen, Ida B Thøgersen, Marie V Lukassen, Litten Rossen, Irene Garcia-Ferrer, Tibisay Guevara, Carsten Scavenius, Ernst Meinjohanns, F Xavier Gomis-Ruth, Jan J Enghild

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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.

Original languageEnglish
JournalJournal of Biological Chemistry
Pages (from-to)2271-2287
Number of pages17
Publication statusPublished - 1 Jan 2016


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