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Structural details of dionain-1. The N-terminal part is shown in blue, and the C-terminal part is shown in gray. A, active site configuration of dionain-1 in complex with E-64 covalently linked to Cys 26 . Relevant hydrogen bonds are shown in orange. The initial Fourier map for E-64 is superimposed with the final refined coordinates for the part of E-64 that was modeled. B, the defining residues of the S 2 pocket of dionain-1 result in a spacious cavity due to Ala 139 and Thr 70 . C, surface presentation of the complete substrate binding cleft in dionain-1 with subsite indication and coloring of important charged groups (S 1 : Gln 148 ; S 1 : Cys 66 , Asn 67 , and Asp 164 ; S 2 : Gln 217 ; S 3 : Asn 62 , Asp 63 , and Asn 67 ). D, comparison of the active site cleft (front view) in papain (purple), Cys-EP (green), and dionain-1 (blue). E, location of loop regions with sequence inserts unique to dionain-1 (compared with papain). Loop 1 is a 2-residue insert (Asp 63 -Arg 64 ), located in the loop region from Cys 57 to Cys 66 , and results in a protrusion toward the S 3 -S 4 subsites. Loop 2 is 3-residue insert (Ala 94 -Gly 95 -Gly 96 ) in the N-terminal domain. Loop 3 is a 6-residue insert (Pro 176 -Ser 181 ) in the C-terminal domain that expands an antiparallel -sheet.
Source publication
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 A resolution....
Contexts in source publication
Context 1
... 137 ) and castor bean cysteine protease (Asp 161 contacts Ala 139 and Gly 140 ). The active site Cys 26 is covalently linked to the C2 atom of the epoxy ring of the cysteine protease inhibitor, E-64, which neatly fits into the subsites of the substrate-binding cleft upstream of the cleavage site and is held in place by multiple hydrogen bonds ( Fig. 8A and Table 3). The arrangement is highly similar to the binding mode found in papain and other cysteine proteases of the same family and causes only a minor expansion of the active site to accommodate the proper coordinate chemistry (60, 61). The oxyanion hole is occupied by the carboxylic acid group of E-64, and the leucyl moiety is ...
Context 2
... to accommodate the proper coordinate chemistry (60, 61). The oxyanion hole is occupied by the carboxylic acid group of E-64, and the leucyl moiety is located in the S 2 pocket of dionain-1, where it is held in place by hydrophobic interactions and by backbone hydrogen bonding of the succeeding E-64 amide nitrogen to the carbonyl oxygen of Gly 69 (Fig. 8A). The 4-guanidinobutane moiety of E-64 is not visible in the electron density map (Fig. 8A), most likely due the absence of interactions with the enzyme to restrict its motion. In papain, Tyr 61 and Tyr 67 hold the guanidinium group in place through bonding via the side-chain hydroxyl groups (61); however, dionain-1 differs by having ...
Context 3
... the carboxylic acid group of E-64, and the leucyl moiety is located in the S 2 pocket of dionain-1, where it is held in place by hydrophobic interactions and by backbone hydrogen bonding of the succeeding E-64 amide nitrogen to the carbonyl oxygen of Gly 69 (Fig. 8A). The 4-guanidinobutane moiety of E-64 is not visible in the electron density map (Fig. 8A), most likely due the absence of interactions with the enzyme to restrict its motion. In papain, Tyr 61 and Tyr 67 hold the guanidinium group in place through bonding via the side-chain hydroxyl groups (61); however, dionain-1 differs by having Arg 64 and Thr 70 in the corresponding positions. This arrangement indicates clear ...
Context 4
... of Dionain-1-The nucleophilic attack on the peptide carbonyl group of the P 1 -P 1 scissile bond by the thiolate anion of Cys 26 is guided by the accommodation of the peptide or peptide-like substrate in the subsites of the active site cleft of dionain-1. The unique subsite arrangement is illustrated in Fig. 8C and indicates the electrostatic groups involved in the selectivity and modulation of substrate binding. The S 1 subsite of dionain-1 is a relatively shallow V-shaped groove that primarily provides main chain stabilization, as seen in the E-64 contacts formed here. The substrate library profiling (Fig. 5) indicates that long-chain ...
Context 5
... Loop 1 is a 2-residue insert (Asp 63 -Arg 64 ), located in the loop region from Cys 57 to Cys 66 , and results in a protrusion toward the S 3 -S 4 subsites. Loop 2 is 3-residue insert (Ala 94 -Gly 95 -Gly 96 ) in the N-terminal domain. Loop 3 is a 6-residue insert (Pro 176 -Ser 181 ) in the C-terminal domain that expands an antiparallel -sheet. (Fig. 8D). The volume of the pocket (279 Å 3 ) is larger than in papain (255 Å 3 ) because Ala 139 is situated at the pocket bottom instead of Val 133 (volumes measured by the program CASTp ...
Context 6
... both Cys-EP and ervatamin A, the cavity-bottom residue is also Ala, but the volume of the S 2 subsite is restricted by substitution of Pro 71 (in dionain-1) with Met (Cys-EP) or Phe (ervatamin A). Additionally, the presence of Thr 70 in dionain-1 in place of papain's Tyr 67 widens the substrate-binding cleft from subsite S 3 outward (Fig. 8D). This places dionain-1 as a promiscuous cysteine protease with a spacious S 2 pocket and a large range of possible interactions to accommodate the P 3 substrate residue and beyond. These structural features agree with the substrate library profiling in which large hydrophobic residues preferentially occupy the S 2 site and low ...
Context 7
... 1 and concomitant preference for Lys/Arg in P 3 (Fig. 5D), dionain adequately carries a negatively charged interface by Asn 62 and Asp 63 that could comprise a stabilizing S 3 subsite. In fact, this interface results from a dionain-1-specific 2-residue insertion (Asp 63 -Arg 64 ) that gives rise to a main-chain protrusion toward subsites S 3 -S 4 (Fig. 8E). For the dionain-1 activity observed for substrates filling the S 2 subsite with a Lys/Arg residue (Fig. 5B), the accommodation of the positive charges in S 2 could be explained by the presence of Gln 217 at the top of the pocket, which may act as a hydrogen bond donor. Such stabilization is observed in cathepsin B, which carries an ...
Context 8
... overall globular structure of dionain-1 is highly similar to the papain-like fold differing primarily by the presence of three sequence inserts (Fig. 8E). The largest sequence insert is found from residue 176 to 181 in the C-terminal domain and leads to expansion of an antiparallel -sheet, located on the distal top side of the active site cleft. In Cys-EP, a similar 5-residue insertion is found, but the functional role of this segment remains to be ...
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Citations
... Although the predicted structures do not capture every detail, particularly when considering side chain conformations, we find that they are highly reliable for predicting the overall folds of enzymes belonging to well-known structural classes, including the cysteine proteases used in this activity. This capability was illustrated by the crystal structure of a cysteine protease from D. muscipula (16), which was solved after we predicted its structure (17). Our predicted structure has excellent overall agreement with the experimental one and captures all of the functionally important features of the active site. ...
A major challenge for science educators is teaching foundational concepts while introducing their students to current research. Here we describe an active learning module developed to teach protein structure fundamentals while supporting ongoing research in enzyme discovery. It can be readily implemented in both entry-level and upper-division college biochemistry or biophysics courses. Preactivity lectures introduced fundamentals of protein secondary structure and provided context for the research projects, and a homework assignment familiarized students with 3-dimensional visualization of biomolecules with UCSF Chimera, a free protein structure viewer. The activity is an online survey in which students compare structure elements in papain, a well-characterized cysteine protease from Carica papaya, to novel homologous proteases identified from the genomes of an extremophilic microbe (Halanaerobium praevalens) and 2 carnivorous plants (Drosera capensis and Cephalotus follicularis). Students were then able to identify, with varying levels of accuracy, a number of structural features in cysteine proteases that could expedite the identification of novel or biochemically interesting cysteine proteases for experimental validation in a university laboratory. Student responses to a postactivity survey were largely positive and constructive, describing points in the activity that could be improved and indicating that the activity was an engaging way to learn about protein structure.
... The fluid undergoes energy-dependent acidification soon after prey capture, and this not only enhances prey confinement and increases the prey killing rate by suffocation, but also allows for chemical decomposition of the prey (Bazile et al., 2015;Gilbert et al., 2020). The acidification is important not only for optimal enzyme activity, but also for the autoactivation of some proteases from proenzymes (Athauda et al., 2004;Risør et al., 2016). Enzymatic decomposition is achieved by a cocktail of hydrolases that are very stable, possess broad substrate specificity, resist proteases and act in acidic conditions. ...
Background
Carnivorous plants are an ecological group of ca. 810 vascular species which capture and digest animal prey, absorb prey-derived nutrients and utilize them to enhance their growth and development. Extant carnivorous plants have evolved in at least ten independent lineages and their adaptive traits represent an example of structural and functional convergence. Plant carnivory is a result of complex adaptations to mostly nutrient-poor, wet and sunny habitats when the benefits of carnivory exceed the costs. With a boost in interest and extensive research in recent years, many aspects of these adaptations have been clarified (at least partly), but many remain unknown.
Scope
We provide some recentmost insights into substantial ecophysiological, biochemical and evolutional particulars of plant carnivory from the functional viewpoint. We focus on those processes and traits in carnivorous plants associated with their ecological characterization, mineral nutrition, cost-benefit relationships, functioning of digestive enzymes and regulation of the hunting cycle in traps. We elucidate mechanisms by which uptake of prey-derived nutrients leads to stimulation of photosynthesis and root nutrient uptake.
Conclusions
Utilization of prey-derived mineral (mainly N and P) and organic nutrients is highly beneficial for plants and increases the photosynthetic rate in leaves as a prerequisite for faster plant growth. Whole-genome and tandem gene duplications brought gene material for diversification into carnivorous functions and enabled recruitment of defense-related genes. Possible mechanisms for the evolution of digestive enzymes are summarized and a comprehensive picture on the biochemistry and regulation of prey decomposition and prey-derived nutrient uptake is provided.
... All sequences from the Drosera capensis genome [5] and the Dionaea muscipula transcriptome [22] that were previously annotated as coding for MEROPS A1 aspartic proteases using the MAKER-P (v2.31.8) pipeline [23] and a BLAST search against SwissProt (downloaded 8/30/15) and InterProScan [24] were examined for the presence of a PSI. Sequence alignment with the previouslycharacterized PSI from the Arabidopsis thaliana protease APA1_ARATH was used for quality control; proteins that did not contain a full-length PSI were not selected for modeling or further analysis. ...
The Droserasins, aspartic proteases from the carnivorous plant Drosera capensis, contain a 100-residue plant-specific insert (PSI) that is post-translationally cleaved and independently acts as an antimicrobial peptide. PSIs are of interest not only for their inhibition of microbial growth, but also because they modify the size of lipid vesicles and strongly interact with biological membranes. PSIs may therefore be useful for modulating lipid systems in NMR studies of membrane proteins. Here we present the expression and biophysical characterization of the Droserasin 1 PSI (D1 PSI.) This peptide is monomeric in solution and maintains its primarily α -helical secondary structure over a wide range of temperatures and pH values, even under conditions where its three disulfide bonds are reduced. Vesicle fusion assays indicate that the D1 PSI strongly interacts with bacterial and fungal lipids at pH 5 and lower, consistent with the physiological pH of D. capensis mucilage. It binds lipids with a variety of head groups, highlighting its versatility as a potential stabilizer for lipid nanodiscs. Solid-state NMR spectra collected at a field strength of 36 T, using a unique series-connected hybrid magnet, indicate that the peptide is folded and strongly bound to the membrane. Molecular dynamics simulations indicate that the peptide is stable as either a monomer or a dimer in a lipid bilayer. Both the monomer and the dimer allow the passage of water through the membrane, albeit at different rates.
... To study the induction of gene expression in the trap tissue, two corresponding genes of well-characterized proteins from digestive fluid were chosen: the cysteine protease dionain (Schulze et al., 2012;Risør et al. 2016) and chitinase I (Paszota et al., 2014). To determine the effect of anaesthesia on gene expression, we had to find the time point where the induction of gene expression is high. ...
Background and aims:
General anaesthetics are compounds that induce loss of responsiveness to environmental stimuli in animals and humans. The primary site of general anaesthetic action is the nervous system, where anaesthetics inhibit neuronal transmission. Although plants do not have neurons, they generate electrical signals in response to biotic and abiotic stresses. Here, we investigated the effect of the general volatile anaesthetic diethyl ether on the ability to sense potential prey or herbivore attacks in the carnivorous plant Venus flytrap (Dionaea muscipula).
Methods:
We monitored trap movement, electrical signalling, phytohormone accumulation and gene expression in response to the mechanical stimulation of trigger hairs and wounding under diethyl ether treatment.
Key results:
Diethyl ether completely inhibited the generation of action potentials and trap closing reactions, which were easily and rapidly restored when the anaesthetic was removed. Diethyl ether also inhibited the later response: jasmonate (JA) accumulation and expression of JA-responsive genes (cysteine protease dionain and type I chitinase). However, external application of JA bypassed the inhibited action potentials and restored gene expression under diethyl ether anaesthesia, indicating that downstream reactions from JA are not inhibited.
Conclusions:
The Venus flytrap cannot sense prey or a herbivore attack under diethyl ether treatment caused by inhibited action potentials, and jasmonate signalling pathway as consequence.
... Briefly, frozen plant material (20 mg) was homogenized and extracted using 1 mL of ice cold CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. To study the induction of gene expression in the trap tissue, two corresponding genes of well-224 characterized proteins from digestive fluid were chosen: the cysteine protease dionain 225 (Schulze et al., 2012;Risør et al. 2016) and chitinase I (Paszota et al., 2014). To determine 226 the effect of anaesthesia on gene expression, we had to find the time point where the 227 induction of gene expression is high. ...
General anaesthetics are compounds that induce loss of responsiveness to environmental stimuli in animals and humans. The primary site of general anaesthetic action is the nervous system, where anaesthetics inhibit neuronal transmission. Although plants do not have neurons, they generate electrical signals in response to biotic and abiotic stresses. Here, we investigated the effect of the general volatile anaesthetic diethyl ether on the ability to sense potential prey or herbivore attacks in the carnivorous plant Venus flytrap ( Dionaea muscipula ). We monitored trap movement, electrical signalling, phytohormone accumulation and gene expression in response to the mechanical stimulation of trigger hairs and wounding under diethyl ether treatment. Diethyl ether completely inhibited the generation of action potentials and trap closing reactions, which were easily and rapidly restored when the anaesthetic was removed. Diethyl ether also inhibited the later response: jasmonate (JA) accumulation and expression of JA-responsive genes. However, external application of JA bypassed the inhibited action potentials and restored gene expression under diethyl ether anaesthesia, indicating that downstream reactions from JA are not inhibited. Thus, the Venus flytrap cannot sense prey or a herbivore attack under diethyl ether treatment. This is an intriguing parallel to the effect of anaesthesia on animals and humans.
Highlight
Carnivorous plant Venus flytrap ( Dionaea muscipula ) is unresponsive to insect prey or herbivore attack due to impaired electrical and jasmonate signalling under general anaesthesia induced by diethyl ether.
... rmsd = 2.9 Å, 322/446 residues). The structural similarities extend to include other members of the broader C1 papain-like proteases, such as Clostridioides difficile Cwp84 (4D5A, Z = 18.2, rmsd = 2.9 Å, 207/406 residues), 36 Dionaea muscipula dionain-1 (5A24, Z = 17.8, rmsd = 2.8 Å, 197/222 residues), 37 and EP-B2 (2FO5, Z = 17.6, rmsd = 2.8 Å, 194/224 residues) 38 (Fig. S9). Like most papain-like proteases, Pd_dinase prefers hydrophobic amino acids in the P2 position, but unlike the papain-like proteases, Pd_dinase strongly prefers Gly at the P2 position 39 and has no P1 specificity. ...
Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroidetes distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, we termed Pd_dinase, has a sequential di-aminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine-AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine-AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 proteases. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.
... Lately, it also has been found that cysteine protease is the primary protease found in digestive fluid of Dionaea (Venus flytrap). Prey proteins found in the digestive fluid of Dionaea are degraded by cysteine endopeptidases in association with serine carboxypeptidases (Risør et al., 2016). This is highly distinct to the digestive fluids found in Nepenthes and Drosera with aspartic proteases (Athauda et al., 2004). ...
Background
Carnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry.
Methods
This review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap) , Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants) , Cephalotus (Australian pitcher plants) , Genlisea (corkscrew plants) , and Utricularia (bladderworts).
Results
Since the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants.
Discussion
These different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications.
... Lately, it also has been found that cysteine protease is the primary protease found in digestive fluid of Dionaea (Venus flytrap). Prey proteins found in the digestive fluid of Dionaea are degraded by cysteine endopeptidases in association with serine carboxypeptidases (Risør et al., 2016). This is highly distinct to the digestive fluids found in Nepenthes and Drosera with aspartic proteases (Athauda et al., 2004). ...
Background. Carnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry.
Methods. This review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap) , Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants) , Cephalotus (Australian pitcher plants) , Genlisea (corkscrew plants) , and Utricularia (bladderworts).
Results. Since the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants.
Discussion. These different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications.
... 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. 2). ...
... (encoded by the gene annotated by an arrow in A; based on the Venus flytrap [D. muscipula] enzyme structure(77)) shows that some residues under positive selection lie within or near the substrate-binding cleft. The cleft is depicted in yellow, and amino acid sites identified as under positive selection are indicated in red or cyan. ...
Significance
Carnivorous plants capture and digest animal prey for nutrition. In addition to being carnivorous, the humped bladderwort plant, Utricularia gibba , has the smallest reliably assembled flowering plant genome. We generated an updated genome assembly based on single-molecule sequencing to address questions regarding the bladderwort’s genome adaptive landscape. Among encoded genes, we segregated those that could be confidently distinguished as having derived from small-scale versus whole-genome duplication processes and showed that conspicuous expansions of gene families useful for prey trapping and processing derived mainly from localized duplication events. Such small-scale, tandem duplicates are therefore revealed as essential elements in the bladderwort’s carnivorous adaptation.
... Our modeling approach, in which the starting Rosetta structures are subjected to in silico maturation, was previously validated experimentally when the X-ray structure of a cysteine pro- tease we had previously predicted was solved. The crystal structure of Dionain 1 (PDB ID: 5A24) [40], shows excellent agreement with our predicted structure, with the prediction capturing all major sec- ondary structural elements and exhibiting only minor deviations in the flexible loop regions [19]. For the chitinases, fragment homology was the primary method used. ...
