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J Biol Chem. 2016 Jan 29;291(5):2271-87. doi: 10.1074/jbc.M115.672550. Epub 2015 Dec 1.

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

Author information

1
From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark, the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark.
2
the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark.
3
From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
4
the Proteolysis Laboratory, Department of Structural Biology ("María de Maeztu" Unit of Excellence), Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona, Catalonia, Spain, and.
5
DuPont Industrial Biosciences, DK-8220 Brabrand, Denmark.
6
From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark, the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark, jje@mbg.au.dk.

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.

KEYWORDS:

Venus flytrap; cysteine proteases; digestion; enzyme; enzyme structure; papain; plant biochemistry; plant carnivory; plant physiology; proteinase

PMID:
26627834
PMCID:
PMC4732211
DOI:
10.1074/jbc.M115.672550
[Indexed for MEDLINE]
Free PMC Article

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