title: Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.
abstract: Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin.
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Paulina
authors.initials: P
authors.lastname: Paszota
authors.affiliation: Institute for Molecular Plant Physiology and Biophysics, Biocenter, University Wuerzburg, D-97070 Wuerzburg, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Escalante-Perez
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Line R
authors.initials: LR
authors.lastname: Thomsen
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Risør
authors.affiliation: Institute for Molecular Plant Physiology and Biophysics, Biocenter, University Wuerzburg, D-97070 Wuerzburg, Germany.
authors.forename: Alicja
authors.initials: A
authors.lastname: Dembski
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Laura
authors.initials: L
authors.lastname: Sanglas
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Tania A
authors.initials: TA
authors.lastname: Nielsen
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Karring
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Ida B
authors.initials: IB
authors.lastname: Thøgersen
authors.affiliation: Institute for Molecular Plant Physiology and Biophysics, Biocenter, University Wuerzburg, D-97070 Wuerzburg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hedrich
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: Institute for Molecular Plant Physiology and Biophysics, Biocenter, University Wuerzburg, D-97070 Wuerzburg, Germany. Electronic address: ifuchs@botanik.uni-wuerzburg.de.
authors.forename: Ines
authors.initials: I
authors.lastname: Kreuzer
authors.affiliation: Department of Molecular Biology and Genetics, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus C, Denmark. Electronic address: krs@mb.au.d.
authors.forename: Kristian W
authors.initials: KW
authors.lastname: Sanggaard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001181:Arthropods / Q000502:physiology*; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004063:Digestion; D031257:Droseraceae / Q000201:enzymology* / Q000235:genetics; D020387:Food Chain; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia; D017433:Protein Structure, Secondary
chemical_list: D002686:Chitin; D002688:Chitinases
keywords: 12-oxo-phytodienoic acid and 4-MU, 4-Methylumbelliferyl; CM-Chitin-RBV; Carboxymethyl-Chitin-Remazol Brilliant Violet; Carnivorous plants; Chitin; Chitinases; DTT; Digestive enzymes; Dionaea muscipula actin transcript; DmACT; GlcNAc; MALDI; N-acetyl-glucosamine; OPDA; Plant biology; dithiothreitol; matrix-assisted laser desorption ionization