title: 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 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.
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark, the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Risør
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark, the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark.
authors.forename: Line R
authors.initials: LR
authors.lastname: Thomsen
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark, the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark.
authors.forename: Kristian W
authors.initials: KW
authors.lastname: Sanggaard
authors.affiliation: the Interdisciplinary Nanoscience Center (iNANO), DK-8000 Aarhus, Denmark.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Nielsen
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Ida B
authors.initials: IB
authors.lastname: Thøgersen
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Marie V
authors.initials: MV
authors.lastname: Lukassen
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Litten
authors.initials: L
authors.lastname: Rossen
authors.affiliation: 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.
authors.forename: Irene
authors.initials: I
authors.lastname: Garcia-Ferrer
authors.affiliation: 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.
authors.forename: Tibisay
authors.initials: T
authors.lastname: Guevara
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.affiliation: DuPont Industrial Biosciences, DK-8220 Brabrand, Denmark.
authors.forename: Ernst
authors.initials: E
authors.lastname: Meinjohanns
authors.affiliation: 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.
authors.forename: F Xavier
authors.initials: FX
authors.lastname: Gomis-Rüth
authors.affiliation: 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.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
mesh_terms: D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D057056:Cysteine Proteases / Q000737:chemistry*; D031257:Droseraceae / Q000201:enzymology*; D004331:Drosophila melanogaster; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D010206:Papain / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D053719:Tandem Mass Spectrometry
chemical_list: D002364:Caseins; D010940:Plant Proteins; D057056:Cysteine Proteases; D003546:Cysteine Endopeptidases; D010206:Papain; D007930:Leucine; D008239:Lysine; C024974:E 64
keywords: Venus flytrap; cysteine proteases; digestion; enzyme; enzyme structure; papain; plant biochemistry; plant carnivory; plant physiology; proteinase