INTRODUCTION
Tomato (Lycopersicon esculentum Mill.) is one of the most widespread horticultural crops in the world. Tomatoes are essential and widely used as raw materials for the food industry, as cooking ingredients, or fresh fruit. Various factors have constrained the growth and tomato yield.
One of them is Fusarium wilt which can devastate tomatoes plant. Fusarium wilt is caused by the fungal pathogen named Fusarium oxysporum f.sp.
lycopersici (FOL). As a soil-borne pathogen, FOL can attack plant roots and damage vascular tissue.
This pathogen can also survive for a very long time in the form of chlamydospores. Because of this fungal structure, FOL can survive in the soil for up to 10 years, even without a host (Chitwood-Brown et al., 2021). A study from McGovern (2015) explained that FOL causes significant losses, with corrected economic losses reaching up to 45%.
To make a devastating incident in the field, first, FOL needs to successfully penetrate plant root tissue and causes wilt. During penetration, extracellular degradative enzymes, such as cutinase are critical to dismantle primary hyphae. As ARTICLE INFO
Keywords:
Cutinase
Fusarium oxysporum f.sp.
lycopersici (FOL) Molecular docking Turmeric
Article History:
Received: October 20, 2022 Accepted: October 18, 2023
*) Corresponding author:
E-mail: [email protected]
ABSTRACT
Fusarium oxysporum f.sp. lycopersici (FOL) is a significant threat to tomato plants with its cutinase enzyme playing a critical role in the penetration into plant root tissue. Addressing this issue requires the gathering of essential data. Molecular docking was used to see the interaction between ligand and cutinase structure, whereas the in vitro evaluation used the rhodamine B medium to see fluorescence light expression as an indicator of cutinase activity. The results of both methods revealed turmeric’s ability can disturb cutinase enzyme and its activity. Molecular docking revealed the type and value of binding interactions between turmeric’s natural components and the cutinase enzyme. This virtual simulation attempted to hypothesize that the ligands from turmeric could obstruct the active site of the cutinase structure in order to degrade the plant cutin polymer. The selected turmeric molecules were bisdemethoxycurcumin, calebin A, curcumin, and demethoxycurcumin, which had shown the ability to bind to the active site of cutinase, namely serine (Ser-142). After docking, these four compounds showed the lowest scores (negative), which indicated an efficient binding of the active site. Experiments were continued in the petri dishes and suggested that turmeric extract successfully expressed the fade of fluorescence light expression.
ISSN: 0126-0537Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia, Decree No: 30/E/KPT/2018
Cite this as: Taruna, A., Khairunnisa', A., Dewi, R. R., Hadiwijoyo, E., Yulianah, I., Syib'li, M. A., & Abadi, A. L. (2023).
Molecular docking and in vitro study revealed the inhibition mechanism of cutinase of Fusarium oxsyporum f.sp lycopersici by natural compounds of local turmeric in Indonesia. AGRIVITA Journal of Agricultural Science, 45(3), 554–569. http://doi.org/10.17503/agrivita.v45i3.3966
Molecular Docking and In Vitro Study Revealed the Inhibition Mechanism of Cutinase of Fusarium oxsyporum f.sp lycopersici by Natural Compounds of Local Turmeric in Indonesia
Ardiyan Taruna1), Amalia Khairunnisa’1), Rifani Rusiana Dewi1), Erekso Hadiwijoyo2), Izmi Yulianah3), Muhammad Akhid Syib’li4*), and Abdul Latief Abadi4)
1) Graduate School of Plant Pathology, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
2) Department of Soil Science, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
3) Department of Agronomy, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
4) Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia
the smallest enzyme of the α/β-hydrolase member (Degani, 2015), cutinase can effectively hydrolyze cutin within the cuticle. Cutin is the composer of plant cuticles that protects the plant from physical damage, like early penetration of pathogenic fungi.
Initially, the pathogen secretes a particular amount of cutinase enzyme to break down the cutin polymer into cutin monomers (Serrano et al., 2014). Thereby, cutinase has a significant role in assisting fungi to enter plant tissues (Manikandan et al., 2018).
Following penetration, FOL establishes itself and invades the plant vascular system (Adhikari et al., 2020).
The cutinase has an active site known as the catalytic triad, consisting of the amino acids serine (Ser-142), aspartate (Asp-197), and histidine (His- 210). This active site plays in binding cutin substrates and through hydrolytic reactions, the polymers cutin can be broken down into monomers cutin (Chen et al., 2013). Therefore, the active site of this enzyme potentially can be disrupted by small molecules (ligands). To investigate the inhibition mechanism, molecular docking is a virtual approach that can elaborate more interaction data between ligand and cutinase (Dar & Mir, 2017). Also, this method can be used to predict optimal binding orientation between ligand and target enzyme (Salmaso & Moro, 2018).
Information related to ligands can be obtained from native plants such as turmeric which has promising compounds to be used in this simulation.
Turmeric (Curcuma longa) is abundant and widely cultivated in Indonesia. Despite its extensive use, numerous studies have looked into its potential as an antifungal agent. Numerous studies have suggested that turmeric extract can prevent the formation of fungi. The study of Chen et al. (2018) demonstrated that turmeric extract effectively inhibits the growth of fungal pathogens, including F. graminearum, F. oxysporum, F. chlamydospores, F. tricinctum, F. culmorum, Alternaria alternate, Sclerotinia sclerotiorum, Botrytis cinerea, Rhizopus oryzae, Cladosporium cladosporioides, and Colletotrichum higginsianum. However, there still needs to be more thorough studies regarding how turmeric inhibits the secreted enzyme of FOL, which is essential for penetration. Therefore, besides in silico, this research also tries to comprehend the inhibitory mechanism using in vitro techniques.
Hence, the study aims to use a molecular docking methodology to evaluate any potential binding interactions between the chemicals found in
turmeric and cutinase and use in vitro techniques to monitor the inhibition of the cutinase activity.
MATERIALS AND METHODS Place and Time
The research was conducted at the Department of Plant Pests and Diseases of Universitas Brawijaya’s Biological Control Laboratory from February 2022 to September 2022.
Sample Collection
There were 49 natural turmeric compounds analyzed by Suprihatin et al. (2020) that were used as ligands. The structure of the ligand has been obtained from the PubChem database (https://pubchem.ncbi.nih.gov/). All ligands were downloaded in sdf format for further molecular docking simulation. The cutinase enzyme was selected as the target protein. The structure of cutinase was obtained from predictive modeling by I-TASSER and downloaded in pdb format.
The active site information was obtained UniProt, namely Ser-142, Asp-197, and His-210 (https://
www.uniprot.org/uniprotkb/A0A0D2YA67/entry).
Structural Modeling of Cutinase Enzyme
The method to construct the structural modeling of cutinase was based on a protocol conducted by Roy et al. (2010), using I-TASSER webserver. This platform can predict enzyme structure and function. The amino acid sequence of cutinase for structural modeling was obtained from UniProt (entry code: A0A0D2YA67). I-TASSER predicted five cutinase structural models, and these data files were created in pdb format. Each available model had a confidence score (C-Score) with value ranges from -5 until 2. The higher C-Score value of the cutinase model was better and more reliable.
Molecular Docking
Molecular docking between ligands and protein receptors was conducted based on Dallakyan
& Olson (2015), simulated using vina wizard on PyRx 0.8 software. All ligands were inputted into the open babel feature in the sdf format (Source from PubChem). Ligand energies were minimized with the Universal Force Field (UFF) parameter in PyRx software. The ligands were converted into pdbqt format using Convert All to Autodock Ligand.
Docking was carried out by specific docking, with the cutinase active site (Ser-142, His-197 and Asp- 210) as the target. The dimensions of the grid box
used were 25 x 25 x 25 A⁰, which placed the active site in the grid box. Nine ligand poses were obtained from the docking results containing binding affinity and RMSD values. The best pose for each ligand was selected and saved in sdf format for interaction analysis. The interaction will be depicted in 3D images using PyMOL2 software and 2D forms using the poseview (https://proteins.plus).
Fungal Isolate
The isolate of fungus F21 was obtained from the collection of the Biological Control Laboratory, Department of Plant Pests and Diseases, Universitas Brawijaya. The fungus was stored with filter paper technique in the -20 0 C freezer. The fungus was cultured on potato dextrose agar (PDA), incubated for seven days at room temperature (±28
0C), and used for identification and in vitro assay.
Morphological Identification of Fungi
Macroscopic and microscopic observations carried out morphological identification. Macroscopic observations, including the shape and color of the colonies, were carried out by growing the isolates on a PDA medium for seven days. Microscopic observations, including the shape and size of the conidia, were observed using the slide culture technique and captured under a scanning electron microscope (SEM: Hitachi TM3000 SEM). SEM observations were conducted at the Bioscience Laboratory, Universitas Brawijaya (East Java, Indonesia).
Molecular Identification of Fungi
Identification of the species isolate fungus was obtained by PCR assay at the genetics laboratory of PT. Genetica Science Indonesia (Tangerang, Indonesia). The molecular identification steps started with DNA extraction, PCR amplification, purification of PCR products, and sequencing (Nurhalimah et al., 2022). DNA was extracted using the quick DNA fungal miniprep kit method (Zymo Research, D6005). DNA extraction was amplified by PCR using universal primer, ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). The PCR condition was set for initial denaturation at 95ºC for 3 minutes, 35 cycles of denaturation at 95ºC for 15 seconds, then annealing at 52ºC for 30 seconds, and extension at 72ºC for 45 seconds, and the final extension of one cycle at 72ºC for 3 minutes. The
results of PCR were checked by electrophoresis with 0.8% TBE agarose and then sequenced to determine the base sequence. The sequencing data were analyzed with BLAST (Basic Local Alignment Search Tool) to determine the base sequence similarity with all sequences in the NCBI GenBank database.
Pathogenicity Test
A pathogenicity test was carried out on tomato seeds planted in plastic pots filled sterilized soil. The isolate of fungus were propagated by placing agar pieces from fungal colonies on autoclaved steamed corn powder and incubated for four weeks (Afifah et al., 2022). Inoculation of FOL is carried out by placing of FOL inoculum into the soil (Güler Güney
& Güldür, 2018). The corn medium was used as an inoculum by mixing 20 grams of inoculum FOL into a plastic pot, while the control was not inoculated.
Each plastic pot was planted with five tomato seeds and planted for three weeks.
Preparation of Turmeric Extract
Turmeric was extracted based on Therasme et al. (2018), using the hot water extraction (HWE) method. Turmeric extract was made with 200 grams of fresh turmeric and 700 ml of aquadest. Turmeric is cut into small pieces and boiled using aquadest.
The boiled water was filtered to take the extract.
The turmeric extract is then poured into a glass media bottle and sterilized for further use in the in vitro tests.
Cutinase Activity Inhibition Assay
Dry weight was recorded firstly from three discs The medium test that was used to see cutinase activity was the rhodamine B medium. This medium was made with a modification of 0.015 grams of rhodamine B, and 1 ml of olive oil was added to 100 ml of PDA media (Panuthai et al., 2012). Inhibition of cutinase activity was measured qualitatively using different concentrations of fresh turmeric extract. The treatments compromise 0%, 1%, and 2% turmeric extract in the medium test. The medium test was inoculated with fungi and incubated for three weeks. The cutinase activity of the isolate was observed under UV light (Bio-Rad Gel Doc XR+
Molecular Imager). A halo fluorescence around the fungal colonies indicated the cutinase activity. This method refers to Rocha et al. (2008), who used halo fluorescence to compare the cutinase enzyme activity on the fungal pathogen F. oxysporum.
RESULTS AND DISCUSSION Structural Modeling
I-TASSER generated the top 5 predictive models’ structures for cutinase enzyme. Each model has a different confident score (C-score). The model with higher values for C-score (model 1, C-score:
0.46, TM-score: 0.77) was selected as a final model for the following analysis. The first model (model 1) generated by I-TASSER is based on the whole-chain model and has better quality than the other models.
The model with C-score >-1.5 and TM-score >0.5 has a correct fold and sound quality (Yang & Zhang, 2015). The selected model was visualized through PyMOL software (Fig. 1A). The three active sites in the model structure have been shown on the stick (Fig. 1B). The selected modelrevealed that the structural model composed of alpha-helix (13%), beta-strand (35%), and coil (52%).
In silico: Screening Ligands and Results Docking
Forty-nine turmeric compounds were virtually screened using PyRx software to assess their ability to bind the active site of cutinase of FOL. Based on the outcomes of the virtual screening, each ligand- enzyme interaction has a different docking score, and all have negative docking scores (Table 1). The most efficient enzyme inhibitors are those with the
lowest docking scores (Mangat et al., 2022). The lowest docking score explains that the ligand only needs minimum energy to bind the active site of cutinase with stable interaction. However, not all ligands from turmeric can interact with enzymes due to their inability to attach to the amino acids on the cutinase within the generated grid box during virtual screening. Poseview (https://proteins.plus) was used to examine the type of interactions between the ligands and enzymes.
Interaction Ligands and Enzyme
Interaction between ligands and enzyme with lower docking scores are more stable during their engagement (Wargasetia et al., 2021). Six particular ligands were selected from the results of the molecular docking tests since they had the lowest docking scores and showed interactions.
These interactions were subsequently visualized to illustrate their binding to the cutinase enzyme (as depicted in Fig. 2A and Fig. 2B). The ligands in focus are bisdemethoxycurcumin (14), calebin A (15), curcumin (17), demethoxycurcumin (26), letestuianin A (33), and quercetin (40). The study delineated three distinct interaction types between the ligands and the cutinase receptor: they were hydrogen bonding, hydrophobic contacts, and pi-pi stacking (as shown in Fig. 2 and Fig. 3) (Stierand &
Rarey, 2010).
Remarks: (A) 3D structure surface; (B) 3D structure cartoon, active sites showed in stick
Fig. 1. Predicted structural modeling of the cutinase using I-TASSER and visualized through the PyMOL2
Remarks: Three-dimensional and two-dimensional visualization of bisdemethoxycurcumin (A and B), calebin A (C and D), and curcumin (E and F) in the binding site of cutinase, repectively. Ligands are showed as green stick and cutinase residues are showed as pink sticks. Hydrogen bonding, represented by a dotted black line (1 arrowhead); hydrophobic contacts, indicated by a green line (2 arrowheads); and pi-pi stacking, denoted by a dotted green line (3 arrowheads).
Fig. 2A. Interaction between selected ligands from turmeric and cutinase
Remarks: Three-dimensional and two-dimensional visualization of bisdemethoxycurcumin (A and B), calebin A (C and D), and curcumin (E and F) in the binding site of cutinase, repectively. Ligands are showed as green stick and cutinase residues are showed as pink sticks. Hydrogen bonding, represented by a dotted black line (1 arrowhead); hydrophobic contacts, indicated by a green line (2 arrowheads); and pi-pi stacking, denoted by a dotted green line (3 arrowheads).
Fig. 2A. Interaction between selected ligands from turmeric and cutinase
Remarks: Three-dimensional and two-dimensional visualization of demethoxycurcumin (A and B), letestuianin A (C and D), Quercetine (E and F) in the binding site of cutinase, repectively. Ligands are showed as green stick and cutinase residues are showed as pink sticks. Hydrogen bonding, represented by a dotted black line (1 arrowhead); hydrophobic contacts, indicated by a green line (2 arrowheads); and pi-pi stacking, denoted by a dotted green line (3 arrowheads).
Fig. 2B. Interaction between selected ligands from turmeric and cutinase
Remarks: Three-dimensional and two-dimensional visualization of demethoxycurcumin (A and B), letestuianin A (C and D), Quercetine (E and F) in the binding site of cutinase, repectively. Ligands are showed as green stick and cutinase residues are showed as pink sticks. Hydrogen bonding, represented by a dotted black line (1 arrowhead); hydrophobic contacts, indicated by a green line (2 arrowheads); and pi-pi stacking, denoted by a dotted green line (3 arrowheads).
Fig. 2B. Interaction between selected ligands from turmeric and cutinase
Table 1. Docking results and interactions of turmeric compounds on cutinase
No. Compounds PubChem
ID
Docking score
(kcal/mol) Type of Interactions 1. 3-Cyclohexene-1,2-diol 12407016 -3.9 H-bond: Thr-145
2. 3-Heptanone 7802 -3.3 No interactions
3. Alpha - Atlantone 12299867 -6.0 No interactions 4. Alpha - Curcumene 92139 -4.7 No interactions 5. Alpha - Turmerone 14632996 -5.2 No interactions
6. Ar -Turmerone 160512 -5.5 No interactions
7. Ascorbic acid 54670067 -4.8 H-bond: Gly-140
8. Beta - Carotene 5280489 -6.4 No interactions
9. Beta - Caryophyllene 5281515 -5.0 No interactions 10. Sesquiphellandrene 519764 -4.8 No interactions 11. Beta - Sitosterol 222284 -6.5 No interactions
12. Bisacumol 5315469 -5.3 H-bond: Tyr-231
Hydrophobic contact: Ala-167, Ala-139, Ala-217
13. Bisacurone 14287397 -5.7 H-bond: Gly-140
14. Bisdemethoxycurcumin 5315472 -6.8 H-bond: Ser-142
Hydrophobic contact: Phe-169, Gly-170
15. Calebin A 637429 -6.8 H-bond: Phe-205
Hydrophobic contact: Ser-142
16. Camphene 6616 -4.3 No interactions
17. Curcumin 969516 -7.0 H-bond: Ser-142, Tyr-61, Phe-205, dan Leu-194 Hydrophobic contact: Val-75, Val-199
18. Cinnamic acid 444539 -4.8 No interactions
19. Curcumenol 167812 -6.2 H-bond: Gly-140
Hydrophobic contact: Ala-217, Tyr-213
20. Curcumenone 153845 -5.6 No interactions
21. Curcumol 14240392 -5.5 H-bond: Gly-140, Ala-167
Hydrophobic contact:Tyr-213
22. Curdione 6441391 -6.0 No interactions
23. Curlone 196216 -5.4 No interactions
24. Curzerenone 3081930 -5.6 pi-pi stacking: Tyr-213 25. Dehydrocurdione 6442617 -5.6 No interactions
26. Demethoxycurcumin 5469424 -7.1 H-bond: Ser-142, Gly-170
Hydrophobic contact: Gly-170, Val-199
27. Caffeic acid 689043 -5.5 H-bond: Val-75
Hydrophobic contact: Ala-217 28. D - Fructose 2723872 -5.2 H-bond: Leu-71, Ala-62, Thr-74 29. Germacrone 4,5 epoxide 13922652 -5.4 No interactions
30. D - Glucose 5793 -5.0 H-bond: Gly-140, Leu-168, Tyr-213
31. Isoprocurcumenol 14543197 -6.5 H-bond: Gly-140
Hydrophobic contact: Leu-79, Tyr-141
Bisdemethoxycurcumin (14) is an analogue of natural curcumin. Bisdemethoxycurcumin is one of the three major compounds in turmeric, with a content of 3% after curcumin 77% and demethoxycurcumin 17% (Sandur et al., 2007). The docking score of bisdemethoxycurcumin was -6.8 kcal/mol. This compound interacted with H-bonding at Ser-142 and Val-199 residues. This compound also interacted with hydrophobic contact on Val-199 and Gly-170 residues. Calebin A (15) is one of active compounds of turmeric and has antioxidant effect (Mueller et al., 2022). The docking score of calebin A was -6.8 kcal/mol. This compound interacted with H-bonding on Phe-205 residue and hydrophobic contact on Ser-142 residue. While, curcumin (17) is the main dominant compound in turmeric and has Table 1. (continued)
No. Compounds PubChem ID Docking
score
(kcal/mol) Type of Interactions 32. L - Arabinose 439195 -4.7 H-bond: Thr-113, Gln 143, Gly-111
33. Letestuianin A 638190 -6.9 H-bond: Ser-13
Hydrophobic contact: Phe-211 Pi-pi stacking : Phe-211
34. Letestuianin B 10429233 -6.7 Hydrophobic contact: Ala-217, Tyr-213 Pi-pi stacking: Tyr-213
35. Limonene 22311 -4.4 No interactions
36. Linalool 6549 -4.1 H-bond: Ala-167
Hydrophobic contact: Val-75, Ala-217
37. Procurcumadiol 14633011 -7.0 No interactions
38. Procurcumenol 189061 -6.6 H-bond: Leu-168
39. Propanal 527 -2.2 No interactions
40. Quercetin 5280343 -7.5 H-bond: Ala-62, Gly-64, Gly-140
Hydrophobic contact: Ala-217
41. Sabinene 18818 -4.3 No interactions
42. Alpha - pinene 6654 -4.2 No interactions
43. Turmeronol B 10955433 -5.8 H-bond: Gly-140
Hydrophobic contact: Ala-217, Ala-139
44. Zedoarondiol 24834047 -6.7 H-bond: Tyr213
Hydrophobic contact: Tyr-213, Tyr-141, Phe-192
45. Alpha - Terpinene 7462 -4.4 No interactions
46. Alpha - Terpineol 17100 -4.5 H-bond: Ala-167, Gly-140
Hydrophobic contact: A;a-217, Phe-192 47. Alpha - Phellandrene 7460 -4.4 No interactions
48. Beta - Pinene 14896 -4.3 No interactions
49. p - Cymene 7463 -4.3 No interactions
antifungal activity against Fusarium sp. (Oza et al., 2021). The docking score of curcumin with cutinase was -7.0 kcal/mol. Curcumin compound interacted with H-bonding type on Tyr-61, Ser-142, Leu-192 and Phe 205 residues. Other interactions that occur were hydrophobic contact on Val-75 and Val-199 residues.
Demethoxycurcumin (26) is an analogue of curcumin that has the potential to inhibit the ATPase fungal (Dao et al., 2016). The docking score of this compound was -7.1 kcal/mol. This compound had H-bonding interaction with cutinase on Ser-142 and Val-199 residues. This compound also interacted with type hydrophobic contact on Val-199 and Gly- 170 residues. Letestuianin A (33) has three types of interactions, namely H-bonding, hydrophobic
five septa. This fungus also produces microconidia with an oval shape and one septum. Hafizi et al.
(2013) also mentioned, that F. oxysporum has white mycelia like cotton and its macroconidia have 3-5 septa. Fourie et al. (2011) also confirmed that this fungus produces chlamydospores with smooth or rough spore surfaces and are formed singly or in pairs.
Molecular Identification
The results of fungal DNA extraction showed that the DNA concentration value was 78.2 ng/ul. Extracted DNA was amplified by PCR using universal primers ITS1 and ITS4. The DNA amplification (PCR product) was assessed by electrophoresis with 0.8% TBE agarose to see the visualization of DNA bands. The results electrophoresis (Fig. 4) showed a DNA band of 550 bp. The results of sequencing using BLAST was showed that the sequenced have a similarity with Fusarium oxysporum (Accession number:
KU671036.1) with a similarity level of 98.44%.
Phatogenicity Test
Pathogenicity test was carried out with the aim of whether the isolates used could infect tomato plants. The results of the test after three weeks of planted, tomato plants showed symptoms of Fusarium wilt disease on tomato plants in which Fusarium oxysporum (FO) inoculated the plant media. Tomato plants that inoculated with FO caused stunted growth, this was confirmed as one of the symptoms of Fusarium wilt (Singh et al., 2017).
In another experiment, tomato plants exhibited symptoms of yellowing leaves (Fig. 5C) and wilting (Fig. 5D). These symptoms are very similar to those of fusarium wilt caused by Fusarium oxysporum f.sp. lycopersici (FOL); morphologically, the tomato plants wilt and the lower leaves turn yellow (Panno et al., 2021; Srinivas et al., 2019). Tomato plants that were not inoculated with FO had a significantly higher plant height than those inoculated (Fig. 5).
Pathogenicity test confirmed that the isolates used could infect tomato plants with a disease incidence rate of 100%. The results showed that the isolate is caused fusarium wilt disease in tomato seedlings.
Because the FO isolate used can infect tomato plants and the symptoms were similar to those of fusarium wilt caused by FOL, the isolate is indicated as F. oxysporum f.sp. lycopersici (FOL).
contact and pi-pi stacked. H-bonding interactions occur with Ser-13 A. This compound had two interaction, namely hydrophobic contact and pi- pi stacked interactions on Phe-211 residue. The results of the docking score between letestuinanin A and cutinase were -6.9 kcal/mol. Quercetin (40) is a flavonoid compound that can reduce the toxicity of mycotoxins (Yang et al., 2020). Quercetin had the best docking score of -7.8 kcal/mol. Quercetin had H-bonding interactions with Gly-64, Ala-62 and Gly- 140 residues. Another interaction that occurs was the type of hydrophobic contact with Ala-217.
From the results of molecular docking, twenty- four from forty-nine compounds had interactions with cutinase, and from six compunds with lowest docking score only four had interactions with the active site of cutinase. The four compounds were bisdemethoxycurcumin, calebin A, curcumin, and demethoxycurcumin interacted with serine (Ser- 142), one of three catalytic triad cutinase members.
These compounds had a higher potential as an inhibitor of cutinase as they could block substrate polymer cutin to bind with catalytic triad cutinase.
All three members of the catalytic triad must bind the polymer cutin to activate of hydrolytic reaction of cutinase. If there are members of the catalytic triad that cannot bind polymer cutin, the hydrolytic reaction cannot be activated (Baker & Montclare, 2010).
Morphological Characterization of Fungi
Based on macroscopic observation, fungal colonies had a circular shape with thick mycelia.
The mycelia of isolate fungi F21 had a smooth texture like cotton. The colonies of fungi showed a creamy white color (Fig. 3A). When observed the fungal isolate in the petridish with upside down, there was a pink pigment on the PDA medium (Fig. 3B). Microscopic examination showed that fungal isolate F21 produced macroconidia which had sickle-shaped and hyaline characteristics.
Macroconidia multicellular had three to five septa and measure 29-30 µm with 3-4 µm width (Fig. 3C and Fig. 3D).
These characteristics have similarities with the fungus Fusarium oxysporum described by Tan et al. (2021), F. oxysporum is circular in shape with a white color and when observed under surface is pink white. F. oxysporum produces macroconidia which are curved and slender and usually have three to
Remakrs: (A) visualization from above petridish (B) visualization from bottom petridish (C) Macroconidia still intact with conidiophore (SEM) (D) Macroconidia has separated from conidiophore (SEM)
Fig. 3. Morphology characterization of Fusarium oxysporum F21
Fig. 4. Visualization of PCR results with ITS1 and ITS4 primers
Fig. 6. Mechanism of formed fluorescence at rhodamine b medium. The halo fluorescence can be detected under uv light.
Remarks: (A) healthy tomato plants (without inoculated FOL), (B) tomato plants was infected Fusarium wilt (with inoculated FO), (C) yellowing, (D) wilting
Fig. 5. Phatogenicity test
In Vitro Assay: Cutinase Activity Inhibition
Cutinase is a hydrolytic enzyme that can be produced by fungi to ease penetrating the plant tissue. To determine cutinase activity, rhodamine b medium was used in this study. Cutinase activity can be detected by the emerge of a halo fluorescence on this medium. The halo fluorescence was formed from an enzyme produced by a fungus that hydrolyzes the triglyceride component of olive oil, releasing free fatty acid acetic acid to form a quaternary ammonium salt. When quaternary ammonium salt interacting with rhodamine b it would formed halo fluorescence (Fig. 6) (Rueda-Rueda et al., 2020).
The test results showed that the addition of turmeric extract caused changes in the visual appearance of the halo fluorescence formed (Fig.
7). There was a reduction in cutinase activity at each concentration level of turmeric extract in petri dishes. Visual observation was used to assess and compare the inhibition of FOL cutinase activity by turmeric extract with different concentrations. At the concentration level of 1% there was a decrease in the halo fluorescence on rhodamine b media compared with control. The cutinase activity was furtherly inhibited as indicated by the decrease in the intensity of the halo fluorescence along with the increase in the concentration of turmeric extract.
At the concentration level of 2%, cutinase activity was at the lowest level. These pictures indicated that the compounds contained in turmeric extract reduced cutinase activity in hydrolyzing the olive oil as substrates.
Fig. 7. Cutinase activity in rhodamine b media with the addition of different concentration levels of turmeric extract
FOL ability to penetrate and infect into plant tissues depends on their hydrolytic enzyme activity. FOL infect the plants through cutinase activity which is secreted to degrade the cuticle which is composed of polymer cutin. The hydrolyzed olive oil in the petri dishes by cutinase of FOL (Fig. 6 and Fig. 7; control) with indicator a halo fluorescence is in vitro simulation of the enzyme activity to break down cutin during penetration (Fig. 9A). In the plant, cutin is the original and main substrates that becomes target of cutinase.
The hypothesis is turmeric compounds can interacting and binding to the active site of cutinase using a competitive inhibition mechanism (Budiman et al., 2022). Turmeric compounds can lock the active site of the enzyme so that cutinase is not able to break down the olive oil as targeted substrates (Fig. 8). Therefore, PDA medium was incorporated with turmeric extract and successfully reduced the halo fluorescence appearance (Fig. 7). Four of the six turmeric compounds observed through molecular docking studies, namely bisdemethoxycurcumin, calebin A, curcumin, and demethoxycurcumin could be the most effective and efficient to bind the active site of cutinase, serine (Ser-142). These four compounds caused inactivation on the catalytic triad site to break down polymer cutin to monomer cutin, so that the cutinase could not assist hyphae of FOL to penetrate the plant tissue (Fig. 7, treatments and Fig. 9B).
-uv light -uv light +uv light +uv light
Medium test
Control (0%)
1%
2%
Fig. 7. Cutinase activity in rhodamine b media with the addition of different concentration levels of turmeric extract
FOL ability to penetrate and infect into plant tissues depends on their hydrolytic enzyme activity.
FOL infect the plants through cutinase activity which is secreted to degrade the cuticle which is composed of polymer cutin. The hydrolyzed olive oil in the petri dishes by cutinase of FOL (Fig. 6 and Fig. 7; control) with indicator a halo fluorescence is in vitro simulation of the enzyme activity to break down cutin during penetration (Fig. 8A). In the plant, cutin is the original and main substrates that becomes target of cutinase. The hypothesis is turmeric compounds can interacting and binding to the active site of cutinase using a competitive inhibition mechanism (Budiman et al., 2022).
Turmeric compounds can lock the active site of
the enzyme so that cutinase is not able to break down the olive oil as targeted substrates (Fig. 9).
Therefore, PDA medium was incorporated with turmeric extract and successfully reduced the halo fluorescence appearance (Fig. 7). Four of the six turmeric compounds observed through molecular docking studies, namely bisdemethoxycurcumin, calebin A, curcumin, and demethoxycurcumin could be the most effective and efficient to bind the active site of cutinase, serine (Ser-142). These four compounds caused inactivation on the catalytic triad site to break down polymer cutin to monomer cutin, so that the cutinase could not assist hyphae of FOL to penetrate the plant tissue (Fig. 7, treatments and Fig. 8B).
Remarks: (A) Fungi produced cutinase to hydrolyze the polymer cutin into monomer cutin, (B) turmeric extract caused cutinase inactivation in hydrolyzing polymer cutin
Fig. 8. Mechanism of turmeric extract to protect plant root tissue
Fig. 9. Mechanism of cutinase activity inhibition by turmeric compounds
The intensity of the halo fluorescence also indicates the amount of enzymes used in hydrolyzing olive oil. The reduction of halo fluorescence in rhodamine B media occurred as much amount of cutinase could not work properly in hydrolyzing olive oil. The higher concentration level of the active compound, the possibility of interaction would be more intense to disturb cutinase enzyme and caused the enzyme lost the hydrolytic abilities (Li et al., 2021; Misas-Villamil & van der Hoorn, 2008). Hence, the halo fluorescence in the petri dish reduced significantly especially at the concentration level of 2%. Further observation until quantitative result of cutinase activity reduction is essential.
CONCLUSION AND SUGGESTION
The molecular docking study of forty-nine natural compounds from turmeric extract identified that twenty-four compounds interacted with cutinase. Four of twenty-four compounds, namely bisdemethoxycurcumin, calebin A, curcumin, and demethoxycurcumin, interacted with the active site of cutinase, specifically serine (Ser-142). These four natural compounds were potentially dominant in reducing the cutinase activity. The molecular docking result was supported by the vitro test that also suggested that the turmeric extract reduced the halo fluorescence of FOL, indicating that high amount of cutinase was not able to break down the targeted substrate. The turmeric extract at the concentration of 2% had the most significant impact in reducing the hydrolizing activity and made halo fluorescence fade away. Hence, quantitative study is essential to investigate the effect of turmeric extract towards cutinase activity of FOL.
ACKNOWLEDGEMENT
We thank to Faculty of Agriculture, Universitas Brawijaya and Hibah Doktor Non Lektor Kepala (DNLK) for the research fund support so that the authors can complete this study and compile the manuscript properly.
REFERENCES
Adhikari, T. B., Gao, A., Ingram, T., & Louws, F. J. (2020).
Pathogenomics characterization of an emerging fungal pathogen, Fusarium oxysporum f. sp.
lycopersici in greenhouse tomato production systems. Frontiers in Microbiology, 11:1995.
https://doi.org/10.3389/fmicb.2020.01995
Afifah, L., Aena, A. C., Saputro, N. W., Kurniati, A., Maryana, R., Lestari, A., Abadi, S., & Enri, U. (2022).
Maize media enhance the conidia production of entomopathogenic fungi Lecanicillium lecanii also its effective to control the weevil Cylas formicarius (Fabricius) (Coleoptera: Brentidae).
AGRIVITA Journal of Agricultural Science, 44(3), 513–525. https://doi.org/10.17503/agrivita.
v44i3.3605
Baker, P. J., & Montclare, J. K. (2010). Biotransformations Using Cutinase. In H. N. Cheng & R. A. Gross (Eds.), ACS Symposium Series (Vol. 1043, pp.
141–158). American Chemical Society. https://
doi.org/10.1021/bk-2010-1043.ch011
Budiman, C., Razak, R. A., Unggit, A. R. A., Razali, R., Suzery, M., Mokhtar, R. A. M., Lee, P.-C., &
Utomo, D. H. (2022). Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide. Molecules, 27(12), 3787.
https://doi.org/10.3390/molecules27123787 Chen, C., Long, L., Zhang, F., Chen, Q., Chen, C., Yu,
X., Liu, Q., Bao, J., & Long, Z. (2018). Antifungal activity, main active components and mechanism of Curcuma longa extract against Fusarium graminearum. PLOS ONE, 13(3), e0194284.
https://doi.org/10.1371/journal.pone.0194284 Chen, S., Su, L., Chen, J., & Wu, J. (2013). Cutinase:
Characteristics, preparation, and application.
Biotechnology Advances, 31(8), 1754–1767.
https://doi.org/10.1016/j.biotechadv.2013.09.005 Chitwood-Brown, J., Vallad, G. E., Lee, T. G., & Hutton,
S. F. (2021). Breeding for resistance to Fusarium wilt of tomato: A review. Genes, 12(11), 1673.
https://doi.org/10.3390/genes12111673
Dallakyan, S., & Olson, A. J. (2015). Small-Molecule Library Screening by Docking with PyRx. In J.
E. Hempel, C. H. Williams, & C. C. Hong (Eds.), Chemical Biology (Vol. 1263, pp. 243–250).
Springer New York. https://doi.org/10.1007/978- 1-4939-2269-7_19
Dao, T. T., Sehgal, P., Tung, T. T., Møller, J. V., Nielsen, J., Palmgren, M., Christensen, S. B., & Fuglsang, A. T. (2016). Demethoxycurcumin Is A Potent Inhibitor of P-Type ATPases from Diverse Kingdoms of Life. PLOS ONE, 11(9), e0163260.
https://doi.org/10.1371/journal.pone.0163260 Dar, A. M., & Mir, S. (2017). Molecular Docking:
Approaches, Types, Applications and Basic Challenges. Journal of Analytical &
Bioanalytical Techniques, 08(02). https://doi.
org/10.4172/2155-9872.1000356
Degani, O. (2015). Production and Purification of Cutinase from Fusarium oxysporum Using Modified Growth Media and a Specific Cutinase Substrate. Advances in Bioscience and Biotechnology, 6(4), 245–258. https://doi.
org/10.4236/abb.2015.64024
Fourie, G., Steenkamp, E. T., Ploetz, R. C., Gordon, T.
R., & Viljoen, A. (2011). Current status of the taxonomic position of Fusarium oxysporum formae specialis cubense within the Fusarium oxysporum complex. Infection, Genetics and Evolution, 11(3), 533–542. https://doi.
org/10.1016/j.meegid.2011.01.012
Güler Güney, İ., & Güldür, E. (2018). Inoculation Techniques for Assessing Pathogenicity of Rhizoctonia solani, Macrophomina phaseolina, Fusarium oxysporum and Fusarium solani on Pepper Seedlings. Türkiye Tarımsal Araştırmalar Dergisi, 5(1), 1–8. https://doi.org/10.19159/
tutad.310211
Hafizi, R., Salleh, B., & Latiffah, Z. (2013). Morphological and molecular characterization of Fusarium.
solani and F. oxysporum associated with crown disease of oil palm. Brazilian Journal of Microbiology, 44(3), 959–968. https://doi.
org/10.1590/S1517-83822013000300047 Li, Y., Wei, J., Yang, H., Dai, J., & Ge, X. (2021). Molecular
dynamics investigation of the interaction between Colletotrichum capsici cutinase and berberine suggested a mechanism for reduced enzyme activity. PLoS ONE, 16(2), e0163260. https://doi.
org/10.1371/journal.pone.0247236
Mangat, H. K., Rani, M., Pathak, R. K., Yadav, I. S., Utreja, D., Chhuneja, P. K., & Chhuneja, P.
(2022). Virtual screening, molecular dynamics and binding energy-MM-PBSA studies of natural compounds to identify potential EcR inhibitors against Bemisia tabaci Gennadius. PLoS ONE, 17(1), e0261545. https://doi.org/10.1371/journal.
pone.0261545
Manikandan, R., Harish, S., Karthikeyan, G., &
Raguchander, T. (2018). Comparative proteomic analysis of different isolates of Fusarium oxysporum f.sp. Lycopersici to exploit the differentially expressed proteins responsible for virulence on tomato plants. Frontiers in Microbiology, 9, 420. https://doi.org/10.3389/
fmicb.2018.00420
McGovern, R. J. (2015). Management of tomato diseases caused by Fusarium oxysporum. Crop Protection, 73, 78–92. https://doi.org/10.1016/j.
cropro.2015.02.021
Misas-Villamil, J. C., & van der Hoorn, R. AL. (2008).
Enzyme-inhibitor interactions at the plant- pathogen interface. Current Opinion in Plant Biology, 11(4), 380–388. https://doi.
org/10.1016/j.pbi.2008.04.007
Mueller, A.-L., Brockmueller, A., Kunnumakkara, A.
B., & Shakibaei, M. (2022). Calebin A, a Compound of Turmeric, Down-Regulates Inflammation in Tenocytes by NF-κB/Scleraxis Signaling. International Journal of Molecular Sciences, 23(3), 1695. https://doi.org/10.3390/
ijms23031695
Nurhalimah, Muhibuddin, A., & Syib’li, M. A. (2022).
Screening of yeast isolates for the biocontrol of Sclerotium rolfsii. Biodiversitas Journal of Biological Diversity, 23(7), 3820–3826. https://
doi.org/10.13057/biodiv/d230759
Oza, K., Khumji, J., & Maitreya, B. (2021). Antifungal Activity of Turmeric (Curcuma longa) Rhizome against Different Fungi. Journal of Natural Sciences and Mathematics, 11(64), 29014–
29017.
Panno, S., Davino, S., Caruso, A. G., Bertacca, S., Crnogorac, A., Mandić, A., Noris, E., & Matić, S. (2021). A review of the most common and economically important diseases that undermine the cultivation of tomato crop in the mediterranean basin. Agronomy, 11(11), 2188.
https://doi.org/10.3390/agronomy11112188 Panuthai, T., Sihanonth, P., Piapukiew, J., Sooksai, S.,
Sangvanich, P., & Karnchanatat, A. (2012).
An extracellular lipase from the endophytic fungi Fusarium oxysporum isolated from the Thai medicinal plant. Croton oblongifolius Roxb. African Journal of Microbiology Research, 6(11), 2622-2638. https://doi.org/10.5897/
ajmr11.965
Rocha, A. L. M., Di Pietro, A., Ruiz-Roldán, C., & Roncero, M. I. G. (2008). Ctf1, a transcriptional activator of cutinase and lipase genes in Fusarium oxysporum is dispensable for virulence.
Molecular Plant Pathology, 9(3), 293–304. https://
doi.org/10.1111/j.1364-3703.2007.00463.x Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: A
unified platform for automated protein structure and function prediction. Nature Protocols, 5(4), 725–738. https://doi.org/10.1038/nprot.2010.5 Rueda-Rueda, H., Prieto-Correa, E., & Jiménez-
Junca, C. (2020). Cutinases obtained from filamentous fungi: A comparison of screening methods. DYNA, 87(214), 183–190. https://doi.
org/10.15446/DYNA.V87N214.83737
Salmaso, V., & Moro, S. (2018). Bridging Molecular Docking to Molecular Dynamics in Exploring Ligand-Protein Recognition Process: An Overview. Frontiers in Pharmacology, 9, 923.
https://doi.org/10.3389/fphar.2018.00923
Sandur, S. K., Pandey, M. K., Sung, B., Ahn, K. S., Murakami, A., Sethi, G., Limtrakul, P., Badmaev, V., & Aggarwal, B. B. (2007). Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-independent mechanism. Carcinogenesis, 28(8), 1765–1773.
https://doi.org/10.1093/carcin/bgm123
Serrano, M., Coluccia, F., Torres, M., L’Haridon, F., &
Métraux, J. P. (2014). The cuticle and plant defense to pathogens. Frontiers in Plant Science, 5(274), 1–8. https://doi.org/10.3389/
fpls.2014.00274
Singh, V. K., Singh, H. B., & Upadhyay, R. S. (2017). Role of fusaric acid in the development of ‘Fusarium wilt’ symptoms in tomato: Physiological, biochemical and proteomic perspectives. Plant Physiology and Biochemistry, 118, 320–332.
https://doi.org/10.1016/j.plaphy.2017.06.028 Srinivas, C., Nirmala Devi, D., Narasimha Murthy, K.,
Mohan, C. D., Lakshmeesha, T. R., Singh, B., Kalagatur, N. K., Niranjana, S. R., Hashem, A., Alqarawi, A. A., Tabassum, B., Abd_Allah, E. F., Chandra Nayaka, S., & Srivastava, R. K.
(2019). Fusarium oxysporum f. sp. lycopersici causal agent of vascular wilt disease of tomato:
Biology to diversity– A review. Saudi Journal of Biological Sciences, 26(7), 1315–1324. https://
doi.org/10.1016/j.sjbs.2019.06.002
Stierand, K., & Rarey, M. (2010). Drawing the PDB:
Protein-ligand complexes in two dimensions.
ACS Medicinal Chemistry Letters, 1(9), 540–
545. https://doi.org/10.1021/ml100164p
Suprihatin, T., Rahayu, S., Rifa, M., & Widyarti, S.
(2020). Senyawa pada Serbuk Rimpang Kunyit (Curcuma longa L.) yang Berpotensi sebagai Antioksidan. Buletin Anatomi dan Fisiologi 5(1), 35–42. https://doi.org/10.14710/baf.5.1.2020.35- 42
Tan, W. J., Thanh, T. A. V., Rafael, E. S., Chen, Y. S., &
Yeo, F. K. S. (2021). Morphological and molecular characterization of Fusarium spp. associated with Fusarium wilt disease of Piper nigrum L.
in Northwestern region of Sarawak. Malaysian Journal of Microbiology, 17(2), 165–177. https://
doi.org/10.21161/mjm.200938
Therasme, O., Volk, T. A., Cabrera, A. M., Eisenbies, M.
H., & Amidon, T. E. (2018). Hot water extraction improves the characteristics of willow and sugar maple biomass with different amount of bark.
Frontiers in Energy Research, 6, 93. https://doi.
org/10.3389/fenrg.2018.00093
Wargasetia, T. L., Ratnawati, H., Widodo, N., &
Widyananda, M. H. (2021). Bioinformatics Study of Sea Cucumber Peptides as Antibreast Cancer Through Inhibiting the Activity of Overexpressed Protein (EGFR, PI3K, AKT1, and CDK4). Cancer Informatics, 20, 117693512110318. https://doi.
org/10.1177/11769351211031864
Yang, D., Wang, T., Long, M., & Li, P. (2020). Quercetin:
Its Main Pharmacological Activity and Potential Application in Clinical Medicine. Oxidative Medicine and Cellular Longevity, 2020, 1–13 https://doi.org/10.1155/2020/8825387
Yang, J., & Zhang, Y. (2015). Protein Structure and Function Prediction Using I‐TASSER. Current Protocols in Bioinformatics, 52(1). https://doi.
org/10.1002/0471250953.bi0508s52
