- Open Access
Homology modeling and docking studies on oxidosqualene cyclases associated with primary and secondary metabolism of Centella asiatica
© Kumar et al.; licensee Springer. 2013
- Received: 27 November 2012
- Accepted: 19 April 2013
- Published: 27 April 2013
Centella asiatica is a well-known medicinal plant, produces large amount of triterpenoid saponins, collectively known as centelloids, with a wide-spectrum of pharmacological applications. Various strategies have been developed for the production of plant secondary metabolites in cell and tissue cultures; one of these is modular metabolic engineering, in which one of the competitive metabolic pathways is selectively suppressed to channelize precursor molecules for the production of desired molecules by another route. In plants the precursor 2,3-oxidosqualene is shared in between two competitive pathways involved with two isoforms of oxidosqualene cyclases. One is primary metabolic route for the synthesis of phytosterol like cycloartenol by cycloartenol synthase; another is secondary metabolic route for the synthesis of triterpenoid like β-amyrin by β-amyrin synthase. The present work is envisaged to evaluate specific negative modulators for cycloartenol synthase, to channelize the precursor molecule for the production of triterpenoids. As there are no experimentally determined structures for these enzymes reported in the literature, we have modeled the protein structures and were docked with a panel of ligands. Of the various modulators tested, ketoconazole has been evaluated as the negative modulator of primary metabolism that inhibits cycloartenol synthase specifically, while showing no interaction with β-amyrin synthase. Amino acid substitution studies confirmed that, ketoconazole is specific modulator for cycloartenol synthase, LYS728 is the key amino acid for the interaction. Our present study is a novel approach for identifying a suitable specific positive modulator for the over production of desired triterpenoid secondary metabolites in the cell cultures of plants.
- Oxidosqualene cyclases
- Cycloartenol synthase
- β-amyrin synthase
- Protein modeling
- Docking studies
Plant natural products and their derivatives play an important role in modern health care as frontline treatments for many diseases and as inspiration for chemical synthesized therapeutics (Pickens et al. 2011). Centella asiatica (L.) Urban, is a well-known medicinal plant, belongs to the family Apiaceae, has tremendous medicinal value and used as an important folk medicinal herb by natives of Asia, southern and middle Africa, southeastern United States and Australia, with a long history of therapeutic uses since ancient times. Preparations of C. asiatica are used in traditional and alternative medicine due to the wide spectrum of pharmacological activities. In common with most traditional phyto-therapeutic agents, C. asiatica is claimed to possess a wide range of pharmacological effects, being used for strengthening the weakened veins (Allegra 1981), wound healing (Sugana et al. 1996), mental disorders (Appa rao et al. 1973), atherosclerosis, fungicidal, antibacterial (Oyedeji & Afolayan 2005), sedative and anxiolytic (Kumar & Gupta 2002), antioxidant and anticancer purposes (Jayashree et al. 2003; Babu et al. 1995), antidepressant (Chen et al. 2003), antiepileptic (Hausen 1993), antinociceptive and anti-inflammatory (Somchit et al. 2004) and radio protective (Sharma & Sharma 2002). C. asiatica has also been reported to be a potent modulator of memory and hunger in both animals and humans, useful in the treatment of venous insufficiency, diarrhea, asthma, fever, improving cognition, tuberculosis and various skin lesions and aliments like leprosy, varicose ulcers, eczema, lupens, psoriasis, diarrhea and keloid (Gohil et al. 2010).
The immense medicinal properties of c. asiatica are attributed to the presence of secondary metabolites known as triterpenoid saponins. The plant contains large amount of triterpenoid saponins, collectively known as centelloids, includes asiaticoside, centelloside, madecassoside, brahmoside, brahminoside, thankuniside, sceffoleoside, centellose, asiatic-, brahmic-, centellic- and madecassic acids. The pharmacological and therapeutic applications of these triterpenes are mainly pentacyclic triterpenic acids and their respective glycosides, belonging to ursane- or oleanane-type, including asiatic acid, asiaticoside, madecassic acid, madecassoside, brahmoside, brahmic acid, brahminoside, thankuniside, isothankuniside, centelloside, madasiatic acid, centic acid, cenellic acid, betulinic acid, indocentic acid etc.
Plant secondary metabolites are incorporated into a wide range of commercial and industrial applications, and fortuitously, in many cases, rigorously controlled plant in vitro cultures can generate valuable natural products. There is great interest in developing alternatives to the intact plant for the production of secondary metabolites. The regular increasing demand in world marketplace for natural and renewable products has focused attention on in vitro plant materials as potential factories for phytochemical products, and has paved the way for new research exploring secondary product expression in vitro. In the recent years new approaches have been developed: the culturing of differentiated cells (e.g. shoots, roots), immobilized cell cultures, hairy root cultures induction by elicitors, tissue engineering and metabolic engineering (Anand 2010; Sevon et al. 1992; Sahai & Knuth 1985; Zhao et al. 2005; Zupan et al. 2000).
One of the approaches by metabolic engineering for the over production of desired metabolite is by blocking the competitive pathways (Verpoorte et al. 1994). Thus, by blocking flow of 2,3-oxidosqualene towards primary metabolism, it is possible to channelize the substrate to secondary metabolism by using suitable modulators that can inhibit primary metabolites-sterol biosynthesis. The aim of the present study is to evaluate and suggest suitable modulators that function like inhibitors for sterol biosynthesis, while without affecting the biosynthesis of triterpenoid secondary metabolites of C. asiatica. To achieve this, in the present study we made an attempt to build the protein structures of cycloartenol synthase (CAS) an enzyme associated with plant sterol (primary metabolite) biosynthesis and β-amyrin synthase (β-AS), an enzyme associated with plant triterpenoid saponin (secondary metabolite) biosynthesis, by homology modeling studies, and also to evaluate the specific interactions of these two enzymes with a panel of modulators by docking studies.
Nucleotide sequences (cDNA) of cycloartenol synthase and β-amyrin synthase of Centella asiatica were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/). These sequences were retrieved into FASTA format and used for further analysis. The modeling of the three dimensional structure of the protein was performed by using SWISS-MODEL (Arnold et al. 2006) (http://swissmodel.expasy.org/), the built model was visualized in molecular visualization software. Structural validation of protein was done using RAMPAGE (Lovell et al. 2002) (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php), phi-psi torsion angles for all the residues in structure were plotted in the Ramachandran Plot at RAMPAGE.
Information regarding modulators of cycloartenol synthase (EC 184.108.40.206) and β-amyrin synthase (EC 220.127.116.11) were retrieved from BRENDA (http://www.brenda-enzymes.org) and also through data mining. These modulators were considered as ligands for the docking studies. Structures of modulators (ligands) were retrieved from PubChem (http://pubchem.ncbi.nlm.nih.gov/) and structures which are not available in the PubChem were drawn in ACD/ChemSketch. All the sdf and mol files obtained from the PubChem and ACD/ChemSketch were converted into pdb files using the Open Babel software. Prediction of ligand binding sites in the modeled protein structure was performed using Q-SiteFinder server (Laurie & Jackson 2005) (http://www.modelling.leeds.ac.uk/qsitefinder/), which were used in docking studies performed in Argus Lab. The modeled and docked structures were visualized in PyMol software.
In order to confirm the significance of LYS 728 in cycloartenol synthase and VAL 728 in β-amyrin synthase reciprocal studies were carried out by amino acid substitutions in the sequences of both cycloartenol synthase and β-amyrin synthase at position 728 residue, wherein cycloartenol synthase LYS 728 was substituted with VAL 728 and in β-amyrin synthase VAL 728 was substituted with LYS 728. Protein structures were modeled and ligand binding studies were carried out in SwissDock (http://www.swissdock.ch/), a free protein ligand docking web service powered by EADock DSS by the Molecular Modeling group of the Swiss Institute of Bioinformatics. The modeled and docked structures were visualized in PyMol software. Similarly modeling of the three dimensional structures both the proteins with substituted amino acid residues at position 728 were carried out using SWISS-MODEL (Arnold et al. 2006) (http://swissmodel.expasy.org/), the built model was visualized in molecular visualization software. Structural validation of protein was done using RAMPAGE (Lovell et al. 2002) (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php), phi-psi torsion angles for all the residues in structure were plotted in the Ramachandran Plot at RAMPAGE.
Retrieved sequences from the databases
Results of protein modeling using SWISS-MODEL
Template PDB ID
Results of structure validation using RAMPAGE
Results of reciprocal studies of protein modeling using SWISS-MODEL
Template PDB ID
Cycloartenol synthase with VAL 728 for LYS 728
β-amyrin synthase with LYS 728 for VAL 728
Results of reciprocal studies of structure validation using RAMPAGE
Cycloartenol synthase with VAL 728 for LYS 728
β-amyrin synthase with LYS 728 for VAL 728
Structure Accuracy with non-substituted structures
Modulators for cycloartenol synthase and β-amyrin synthase
Modulators used in the present study
Predicted ligand binding site of CAS and β-AS using Q-SiteFinder
Docking results of CAS and BAS using Argus Lab
Best ligand pose (E = kcal/mol)
Ketoconazole is an azole fungicide, inhibits both fungal and mammalian cytochrome P450 oxidases (CYPs) that are associated with sterol metabolism. At concentrations >100 nM, ketoconazole inhibits both fungal and mammalian CYP51s, that play an important role in ergosterol and cholesterol biosynthesis respectively, and also affect the activity of enzymes involved in catabolism of cholesterol. More specifically, ketoconazole inhibits 17-hydroxylase-17,20-lyase (CYP17), the cholesterol side chain cleavage enzyme (CYP11A1), and the 11-β-hydroxylase (CYP11B1) (Vanden 1992). The 50% inhibitory concentration (IC50) of ketoconazole for lanosterol synthase was elucidated to be 11.7 nM (Sakaeda et al. 2005). Of the two oxidosqualene cyclases investigated in the present study, cycloartenol synthase is considered to be a plant equivalent for cholesterol synthesis in animals and ergosterol synthesis in fungi. The cyclization is executed with a remarkable degree of specificity and stereochemical control to produce protosterol intermediates. The two ‘protosterols’ that are subsequently modified to functional products such as cholesterol or phytosterols. The products are either lanosterol (in animals and fungi) or cycloartenol (in plants). The two enzymes mediate the cyclization process identically until the final deprotonation step. A deprotonation from C9 forms the 8,9-double bond of lanosterol whereas a deprotonation from C19 allows the cycloartenol cyclopropyl ring to close. Thus far, lanosterol synthase has been found only among the opisthokonts (animals + fungi + choanozoa), trypanosomatids (Trypanosoma, Leishmania) and dinoflagellates (Giner et al. 1991; Roger et al. 2006). All other eukaryotes that have been examined in this regard (at least members of the higher plants, green and red algae, amoebozoa, diatoms, euglenids and heterolobosea) make cycloartenol as their protosterol (Roger et al. 2006). Results of ligand binding site analysis using Q-SiteFinder revealed that, ketoconazole interaction with cycloartenol synthase is hydrophilic interaction with Lys 728 (hydrophilic amino acid) at the active site, whereas it is substituted with Val 728 (hydrophobic amino acid) at the same position in β-amyrin synthase. This hydrophilic-to- hydrophobic substitution of single amino acid residue at the enzyme active site probably distinguishes the two OSC isoforms to show distinctive interaction specificity with the ligand ketoconazole. This has also been proved through reciprocal amino acid substitution studies in both the enzymes.
The in vitro plant enzyme modulator studies are long and expensive one. It starts from target identification, after that, validates the targets and identifies modulators. Due to the limitation of throughput, accuracy and cost, experimental techniques cannot be applied widely; therefore, our study has shifted to in silico approaches such as homology modeling and protein-ligand interactions. In silico approach has been of great importance as a versatile tool to develop fast and accurate target identification and prediction method for the discovery. The present work is an attempt to identify a specific modulator that would control the primary metabolism and over produce the secondary metabolites by channeling the precursor/substrate in Centella asiatica cell cultures because of its immense medicinal importance. The docking studies particularly with ketoconazole has explored the fact that, the two oxidosqualene isoforms differ from each other by virtue of a single amino acid substitution at 728 position from lysine to valine. The results of the present study, suggest that, because of its hydrophilic interaction, ketoconazole can possibly channelize the precursor molecule 2,3-oxidosqualene towards secondary metabolism by functioning like a negative modulator of sterol biosynthesis, and at the same time as a positive modulator for the over production of triterpenoid secondary metabolites in not only cell cultures Centella asiatica of other plants too. To prove this, in our lab we have initiated studies using the cell suspension cultures of Gymnema sylvestre, for the overproduction of gymnemic acid, a group of triterpenoid saponins.
The Financial support from Department of Biotechnology, Ministry of Science and Technology, Government of India (No. BT/PR13872/PID/06/585/2010; Dt.: 30-09-2011) in the form Research Project grant to Vadlapudi Kumar is gratefully acknowledged.
- Abe I, Prestwich GD: Active site mapping of affinity labeled rat oxidosqualene cyclase. J Biol Chem 1994, 269(2):802-804.Google Scholar
- Abe I, Sankawa U, Ebizuka Y: Purification of squalene-2,3-epoxide cyclase from pea seedlings. Chem Pharm Bull 1992, 40(7):1755-1760. 10.1248/cpb.40.1755View ArticleGoogle Scholar
- Abe I, Rohmer M, Prestwich GD: Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem Rev 1993, 93: 2189-2206. 10.1021/cr00022a009View ArticleGoogle Scholar
- Allegra C: Comparative Capillaroscopic study of certain bioflavonoids and total triterpenic fractions of Centella asiatica in venous insufficiency. Clin Ther 1981, 99: 507-513.Google Scholar
- Anand S: Various approaches for secondary metabolite production through plant tissue culture. Pharmacia 2010, 1(1):1-7.Google Scholar
- Appa rao MVR, Srinivasan K, Rao K, et al.: The effect of mandookparni ( Centella asiatica ) on the general mental ability (Medhya) of mentally retarded children. J Res Indian Med 1973, 8: 9-16.Google Scholar
- Arnold K, Bordoli L, Kopp J, Schwede T: The SWISS_MODEL workspace: a web-based environment for protein structure homology modeling. Bioinformatics 2006, 22(2):195-201. 10.1093/bioinformatics/bti770View ArticleGoogle Scholar
- Babu TD, Kuttan G, Padikkala J: Cytotoxic and anti-tumor properties of certain texa of umbelliferae with specific reference to Centella asiatica (L) Urban. J Ethnopharmacol 1995, 48(1):53-57. 10.1016/0378-8741(95)01284-KView ArticleGoogle Scholar
- Beastall GH, Rees HH, Goodwin TW: Properties of 2,3-oxidosqualene – cycloartenol cyclase from Ochromonas malhamensis. FEBS Lett 1971, 18(1):175-178. 10.1016/0014-5793(71)80438-6View ArticleGoogle Scholar
- Chen Y, Han T, Qin L, Rui Y, Zheng H: Effects of total triterpenes of Centella asiatica on the depression behavior and concentration of amino acid in forced swimming mice. Zhong Yao Cai 2003, 26(12):870-873.Google Scholar
- Delprino L, Balliano G, Cattel L, Benveniste P, Bouvier P: Inhibition of higher plant 2,3-oxidosqualene cyclases by 2-aza-2,3-dihydrosqualene and its derivatives. J Chem Soc Chem Commun 1983, 7: 381-382.View ArticleGoogle Scholar
- Fenner GP, Raphiou I: Growth of Cucurbita maxima L. plants in the presence of the cycloartenol synthase inhibitors U18666A. Lipids 1995, 30(3):253-256. 10.1007/BF02537829View ArticleGoogle Scholar
- Flores-Sanchez IJ, Ortega-Lopez J, Montes-Horcasitas MC, Ramos-Valdivia AC: Biosynthesis of sterols and triterpenes in cell suspension culture of Uncaria tomentosa . Plant Cell Physiol 2002, 43(12):1502-1509. 10.1093/pcp/pcf181View ArticleGoogle Scholar
- Giner JL, Wünsche L, Andersen RA, Djerassi C: Dinoflagellates cyclize squalene oxide to lanosterol. Biochem Syst Ecol 1991, 19: 142-145.View ArticleGoogle Scholar
- Gohil KJ, Patel JA, Gajjar AK: Pharmacological review on Centella asiatica : a potential herbal cure-all. Indian J Pharm Sci 2010, 72(5):546-556. 10.4103/0250-474X.78519View ArticleGoogle Scholar
- Goldman RC, Zakula D, Capobianco JO, Sharpe BA, Griffin JH: Inhibition of 2,3-oxidosqualen-lanosterol cyclases in Candida albicans by Pyridinium Ion-based inhibitors. Antimicrob Agents Chemother 1996, 40(4):1044-1047.Google Scholar
- Hausen BM: Centella asiatica (Indian pennywort), an effective therapeutic but a weak sensitizer. Contact Dermatitis 1993, 29(4):175-179. 10.1111/j.1600-0536.1993.tb03532.xView ArticleGoogle Scholar
- Hu X, Neill SJ, Cai W, Tang Z: Nitric oxide mediates elicitor-induced saponin synthesis in cell cultures Panax ginseng . Funct Plant Biol 2003, 30: 901-907. 10.1071/FP03061View ArticleGoogle Scholar
- Jayashree G, Kurup MG, Sudarslal VS, et al.: Antioxidant activity of Centella asiatica on lymphoma bearing mice. Fitoterpia 2003, 74(5):431-434. 10.1016/S0367-326X(03)00121-7View ArticleGoogle Scholar
- Jenner H, Townsend B, Osbourn A: Unravelling triterpene glycoside synthesis in plants: phytochemistry and functional genomics join forces. Planta 2005, 220(4):503-506. 10.1007/s00425-004-1434-zView ArticleGoogle Scholar
- Kim OT, Kim MY, Hwang SJ, Ahn JC, Hwang B: Cloning and molecular analysis of cDNA encoding cycloartenol synthase from Centella asiatica (L.) Urban. Biotechnol Bioprocess Eng 2005, 10: 16-22. 10.1007/BF02931177View ArticleGoogle Scholar
- Kumar MHV, Gupta YK: Effect of different extracts of Centella asiatica on cognition and markers of oxidative stress in rats. J Ethnopharmacol 2002, 79(2):253-260. 10.1016/S0378-8741(01)00394-4View ArticleGoogle Scholar
- Laurie AT, Jackson RM: Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites. Bioinformatics 2005, 21(9):1908-1916. 10.1093/bioinformatics/bti315View ArticleGoogle Scholar
- Lovell SC, Davis IW, Arendall WB III, de Bakker PIW, Word JM, Prisant MG, Richardson JS, Richardson DC: Structure validation by Calpha geometry: phi, psi and Cβ deviation. Proteins 2002, 50(3):437-450.View ArticleGoogle Scholar
- Mangas S, Bonifill M, Osuna L, Moyano E, Tortoriello J, Cusido RM, Pinol MT, Palazom J: The effect of methyl jasmonate on triterpene and sterol metabolism of Centella asiatic , Ruscus aculeatus , and Galphimia glauca cultured plants. Phytochemistry 2006, 67(18):2041-2049. 10.1016/j.phytochem.2006.06.025View ArticleGoogle Scholar
- Oyedeji OA, Afolayan AJ: Chemical composition and antibacterial activity of the essential oil of Centella asiatica growing in South Africa. Pharma Biol 2005, 43(3):249-252. 10.1080/13880200590928843View ArticleGoogle Scholar
- Phillips DR, Rasbery JM, Bartel B, Matsuda SPT: Biosynthetic diversity in plant triterpene cyclization. Curr Opin Plant Biol 2006, 9(3):305-314. 10.1016/j.pbi.2006.03.004View ArticleGoogle Scholar
- Pickens LB, Tang Y, Chooi Y: Metabolic engineering for the production of natural products. Ann Rev Chem Biomol Engg 2011, 2: 211-236. 10.1146/annurev-chembioeng-061010-114209View ArticleGoogle Scholar
- Poralla K, Hewelt A, Prestwich GD, Abe I, Reipen I, Sprenger G: A specific amino acid repeat in squalene and oxidosqualene cyclases. Trends Biochem Sci 1994, 19(4):157-158. 10.1016/0968-0004(94)90276-3View ArticleGoogle Scholar
- Roger ES, Alexander SB, Linda LJ, Jacob RW: Steroids, triterpenoids and molecular oxygen. Philos Trans R Soc Lond B Biol Sci 2006, 361(1470):951-968. 10.1098/rstb.2006.1837View ArticleGoogle Scholar
- Sahai O, Knuth M: Commercializing Plant Tissue Culture. Processes: Economics, Problems and Prospects. Biotechnol Prog 1985, 1(1):1-9. 10.1002/btpr.5420010104View ArticleGoogle Scholar
- Sakaeda T, Iwaki K, Kakumoto M, Nishikawa M, Niwa T, Jin JS, Nakamura T, Nishiguchi K, Okamura N, Okumura K: Effect of micafungin on cytochrome P450 3A4 and multidrug resistance protein 1 activitities, and its comparison with azole antifungal drugs. J Pharm Pharmacol 2005, 57: 759-764. 10.1211/0022357056118View ArticleGoogle Scholar
- Schmitt P, Gonzales R, Benveniste P, Cerutti M, Cattel L: Inhibition of sterol biosynthesis and accumulation of 2,3-oxidosqualene in bramble cell suspension culture treated with 2-aza-2,3-dihydrosqualene and 2-aza-2,3-dihydrosqualene-N-oxide. Phytochemistry 1987, 26: 2709-2714. 10.1016/S0031-9422(00)83577-3View ArticleGoogle Scholar
- Sevon N, Hiltunen R, Oksman-Caldentey KM: Chitosan increases hyoscyamus content in hairy root cultures of Hyoscyamus muticus. Pharm Pharmacol Lett 1992, 2: 96-99.Google Scholar
- Sharma J, Sharma R: Radioprotection of Swiss albino mouse by Centella asiatica extract. Phytother Res 2002, 16(8):785-786. 10.1002/ptr.1069View ArticleGoogle Scholar
- Somchit MN, Sulaiman MR, Zuraini A, Samsuddin LN, Somchit N, Israf DA, et al.: Antinociceptive and antiinflammatory effects of Centella asiatica . Indian J Pharmacol 2004, 36(6):377-380.Google Scholar
- Sugana L, Sivakumar P, Chandrakasan G: Effects of Centella asiatica extract on dermal wound healing in rats. Indian J Exp Biol 1996, 34(12):1208-1211.Google Scholar
- Taton M, Benveniste P, Rahier A: N-[(1,5,9)-Trimethyl-decyl]-4alpha,10-dimethyl-8-aza-trans-decal-3beta-ol a novel potent inhibitor of 2,3-oxidosqualene cycloartenol and lanosterol cyclases. Biochem Biophys Res Commun 1986, 138(2):764-770. 10.1016/S0006-291X(86)80562-9View ArticleGoogle Scholar
- Taton M, Benveniste P, Rahier A, Johnson WS, Liu H, Sudhakar AR: Inhibition of 2,3-oxidosqualene cyclases. Biochemistry 1992, 31(34):7892-7898. 10.1021/bi00149a021View ArticleGoogle Scholar
- Vanden BH: Inhibitors of P450-dependend steroid biosynthesis: from research to medical treatment. J Steroid Biochem 1992, 43: 1003-1021. 10.1016/0960-0760(92)90328-GView ArticleGoogle Scholar
- Verpoorte R, van der Heijden R, Hoge JHC, ten Hoopen HJG: Plant cell biotechnology for the production of secondary metabolites. Pure Appl. Chem 1994, 66(10/11):2307-2310.Google Scholar
- Zhao J, Davis LC, Verpoorte R: Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 2005, 23(4):283-333. 10.1016/j.biotechadv.2005.01.003View ArticleGoogle Scholar
- Zupan J, Muth TR, Draper O, Zambryski P: The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 2000, 23(1):11-28. 10.1046/j.1365-313x.2000.00808.xView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.