Turkish Journal of Biology Turk J Biol (2018) 42: 213-223 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1710-107

In vitro and in silico studies of chalcone synthase variant 2 in rotunda and its substrate specificity

Ragaventhan SANMUGAVELAN, Teow Chong TEOH*, Nurnadiah ROSLAN, Zulqarnain MOHAMED Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

Received: 31.10.2017 Accepted/Published Online: 29.03.2018 Final Version: 13.06.2018

Abstract: In this study, transformation of BrCHS var 2 into B. rotunda cell suspension culture, followed by chalcone synthase enzymatic assay and HPLC analysis was conducted to investigate whether the substrate specificity for BrCHS var 2 is either cinnamoyl-CoA or p-coumaroyl-CoA. The HPLC profile showed an increase in the amount of pinocembrin chalcone when cinnamoyl-CoA and malonyl-CoA were added but not p-coumaroyl-CoA. Molecular docking was performed to explore the binding of cinnamoyl-CoA and p-coumaroyl-CoA to BrCHS var 2 receptor and the docking results showed that cinnamoyl-CoA formed numerous hydrogen bonds and more negative docked energy than p-coumaroyl-CoA. Cinnamoyl-CoA showed good interactions with Cys 164 to initiate the subsequent formation of pinocembrin chalcone, whereas the hydroxyl group of p-coumaroyl-CoA formed an unfavorable interaction with Gln 161 that caused steric hindrance to subsequent formation of naringenin chalcone. Docked conformation analysis results also showed that malonyl-CoA formed hydrogen bonding with Cys 164, His 303, and Asn 336 residues in BrCHS var 2. The results show that cinnamoyl-CoA is the preferred substrate for BrCHS var 2.

Key words: Chalcone synthase, cell suspension culture, homology modelling, molecular docking

1. Introduction The CHS gene constitutes a multigene family in which was used as a food, , and herbal remedy over it has irregular copy number of CHS in different 2000 years ago. It is a monocot plant from the family species. Published studies have reported that only one that can be found widely in . CHS was found in Arabidopsis thalian (Feinbaum and Common members of this family include root ginger Ausubel, 1988) and eight in Sorghum bicolor and Glycine (Zingiber officinale), fingerroot Boesenbergia( rotunda), max, while Vitis vinifera and Physcomitrella patens have (Curcuma longa), and (Zingiber mioga three and seventeen copies, respectively (Goto-Yamamoto Roscoe). There has been increasing interest in its et al., 2002; Lo et al., 2002; Tuteja et al., 2004; Koduri et as a good source of medical treatment for humans and it al., 2010). Initial results on gene isolation from B. rotunda has been utilized as a vital source to exhibit inhibitory CHS (BrCHS) revealed multiple BrCHS variants that were activities as an anticancer, antimicrobial, antiviral, and identified from different parts of the B. rotunda plant. antiinflammatory agent (Sohn et al., 2005; Kiat et al., BrCHS variant 2 (BrCHS var 2) was found to be expressed 2006; Voravuthikunchai et al., 2006; Kirana et al., 2007). predominantly in the rhizome. As such, this variant was In most , chalcone synthase (CHS; EC 2.3.1.74) is chosen to be introduced into B. rotunda cell suspension one of the key enzymes involved in the initiation of the cultures for downstream HPLC analyses. flavonoid biosynthesis pathway. CHS is a plant-specific CHS has broad substrate preference toward aromatic polyketide synthase type III (PKSIII) that forms chalcone and aliphatic CoA esters (Jez et al., 2002; Samappito et by the condensation of one molecule of p-coumaroyl al., 2002; Abe et al., 2007). The first study on evaluating CoA with three molecules of malonyl-CoA to form catalytic activity of the CHS enzyme was performed in metabolites such as an intermediate naringenin chalcone. cell suspension cultures of (Petroselinum hortense) To date, there are more than 2000 nonvolatile compounds (Kreuzaler and Hahlbrock, 1975). p-Coumaroyl-CoA detected using (LCMS) in fresh ginger and rhizome, but was reported to be the most preferential starter molecule less than 100 have been structurally identified (Koo et al., for CHS in many plants as well as cinnamoyl-CoA, 2013). which is catalyzed at a considerable rate as compared to

* Correspondence: [email protected] 213 SANMUGAVELAN et al. / Turk J Biol p-coumaroyl-CoA in few plants (Hatayama et al., 2006). pH 7.6, containing 1 M urea in place of the CHAPS. To Several plants prefer certain substrates and performed a test the CHS enzyme activity, 100 µg of total crude protein typical CHS function such as in Freesia hybrid when using extracted from cell suspension cultures was assayed using p-coumaroyl-CoA and malonyl-CoA as substrates (Sun et two sets of enzymatic reaction; Set 1: 40 µM malonyl- al., 2015). Besides that, CHS from Scutellaria baicalensis CoA (Sigma-Aldrich, USA) with 20 µM cinnamoyl-CoA preferred both aromatic and aliphatic CoA esters such (MicroCombiChem, Germany) and Set 2: 40 µM malonyl- as benzoyl-CoA, phenylacetyl-CoA, isovaleryl-CoA, and CoA (Sigma-Aldrich, USA) with 20 µM p-coumaroyl-CoA isobutyryl-CoA substrates to produce unnatural aromatic (MicroCombiChem, Germany). The reaction mixture was polyketide (Morita et al., 2000). More tests on enzyme prepared in a final volume of 500 µL of 100 mM potassium functionality should be done on the other BrCHS variants phosphate buffer (pH 7.2) containing 0.1% Triton X-100, that can produce diverse metabolites with different incubated at 30 °C for 2 h and stopped by acidification enzymatic function. using 7.5 µL of 1 N HCl. Computational simulations have been used to study The reaction products were partitioned with 800 µL the kinetics and binding modes of drugs (Tsou et al., 2012; of ethyl acetate and concentrated by N2 flow. The residues Shen et al., 2016), homology modelling, and docking of were dissolved in 100 µL of methanol and separated by chalcone synthase from Coleus forskohlii (Awasthi et al., a reversed-phase HPLC system equipped with a Waters 2016). As an added advantage, computational docking 1525 Binary HPLC pump and Waters 2998 photo diode study provides further insight into the possible chemical array detector (Waters, USA) on a Kinetex RP C18 interactions of a ligand to its receptor and hence the (150 mm × 4.6 mm) column (Phenomenex, USA). The elucidation of the ligand–receptor binding mechanism. solvent systems used for the elution were (i) solvent A:

This is the first report on combinatorial in vitro and in CH3OH containing 0.01% H3PO4 and (ii) solvent B: H2O silico studies of one of the CHS of Boesenbergia rotunda by containing 0.01% H3PO4. The gradient elution profile using CHS substrate specificity enzymatic assay, followed consisted of an isocratic step of 50% of solvent A for 1 min, by computational docking verification. The information and a linear gradient from 50% to 100% of solvent A for obtained from this study could also be exploited for 10 min, followed by an isocratic step at 100% of solvent A the future production of novel polyketides using cell for 10 min with a flow rate of 0.8 mL/min. Peak detection suspension cultures. was monitored at 290 nm. Pinocembrin chalcone isolated from B. rotunda (kindly provided by NA Rahman and YK 2. Materials and methods Lee), naringenin, naringenin chalcone, and pinocembrin 2.1. Transformation of BrCHS var 2 into B. rotunda cell (MicroCombiChem, Germany) were used as reference suspension culture compounds. The pGEM-T vector harboring the entire CDS ofBrCHS 2.3. Molecular modelling of ligands and CHS var 2 var 2 was digested with restriction endonucleases, NcoI receptor and SpeI (Fermentas, USA), followed by purification, All the ligand molecular structures were retrieved from ligation into pCAMBIA-1304 (GenBank accession no. PubChem Compound at https://pubchem.ncbi.nlm.nih. AF234300.1), and transformation into Agrobacterium gov/search/search.cgi. The ligand structures used were tumefaciens, strain LBA4404. Positive transformants were cinnamoyl-CoA (CID: 6444037), p-coumaroyl-CoA (CID: selected and subsequently transformed into B. rotunda cell 5462161), and malonyl-CoA (CID: 644066). In addition, suspension culture as described by Yu et al. (2007). After 3 acetyl-CoA (CID: 444439) and Co-A (CID: 46936280) days of co-cultivation, the resistant calli were then selected were docked as references. The ligands were then modelled on semisolid Murashige and Skoog medium containing 300 by Discovery Studio Client v4.5.0.15071 and minimized by mg/L cefotaxime (Duchefa Biochemie, Netherlands) and CHARMm force field (Accelrys Inc., Dassault Systèmes, 15 mg/L hygromycin (Duchefa Biochemie, Netherlands) BIOVIA Corp., San Diego, CA, USA). and were subcultured every 4 weeks. Calli that showed Multiple sequence alignment was carried out by resistance to antibiotics were transferred into liquid media aligning in-house amino acid sequences of BrCHS and subcultured for up to 2 months to get a sufficient var 2 with amino acid sequences from Cucurma number of cells. After 2 months, the cells were harvested longa (ClPKS9; JN017186.1), Curcuma alismatifolia and subjected to protein extraction and HPLC analysis. (CaCHS; GU140082.1), Musa acuminata (MaPKSIII3; 2.2. CHS enzymatic assay and HPLC analysis GU724609.1), Oryza sativa (OsCHS; AB000801.2), Zea The total crude protein was extracted from the transformed mays (ZmCHS; NM_001155550.1), and Medicago sativa and wild-type cell suspension cultures as described by (MsCHS2; L02902.1) using Clustal Omega (https://www. Carpentier et al. (2005) with minor modification. At the ebi.ac.uk/Tools/msa/clustalo/). The homology model last step, the pellet was dissolved in 50 mM Tris-HCl buffer, of BrCHS var 2 receptor was modelled from the in-

214 SANMUGAVELAN et al. / Turk J Biol house amino acid sequence (Supplementary material assay. According to the results, there was an increase in Figure A1) with template sequence of 4YJY (z-score: the amount of pinocembrin chalcone (Figure 1a) when –0.814) (Supplementary material Figure A2) by using the cinnamoyl-CoA and malonyl-CoA were added to the total YASARA Structure modelling software (Krieger et al., crude protein extract. Pinocembrin chalcone (labelled 2002). The stereochemical quality of BrCHS var 2 protein as C1 in Figure 1a) was eluted at a retention time of 8.80 3D receptor model was then checked by PROCHECK min. The estimated amount of C1 compound detected (Laskowski et al., 1993) webserver at https://services.mbi. during HPLC in wild-type and transgenic cell suspension ucla.edu/SAVES/. The homology model was minimized in cultures were 64.67 mg/g (SE: 6.67E-02) and 112.20 GROMACS v5.1.4. (Abraham et al., 2015) initially with mg/g (SE: 1.00E-01), differing significantly at P < 0.05, steepest descent and followed by conjugate gradient to respectively. Cinnamoyl-CoA is another starter molecule energy convergence of 0.01 kJ/mol. that is catalyzed by CHS at comparable rate as compared 2.4. Molecular docking of BrCHS var 2 to p-coumaroyl-CoA in some plants (Christensen et al., The minimized ligands were targeted on the BrCHS var 1998; Fliegmann et al., 1992). 2 receptor binding site containing Cys 164, His 303, CHS has broad substrate specificity (Schüz et al., 1983; and Asn 336 as reported by Jez and Noel (2000) and Jez et al., 2002; Samappito et al., 2002) with p-coumaroyl- confirmed by multiple sequence alignment. The Haddock CoA is the most preferential starter molecule found in 2.2 molecular docking software web server was used to many plants (Yamazaki et al., 2001; Hatayama et al., 2006). perform the docking simulation using the protein–ligand However, it was unpredictably found that BrCHS var 2 was module (de Vries et al., 2010; Wassenaar et al., 2012). The inactive to p-coumaroyl-CoA and no naringenin chalcone lowest docked energy ± standard deviation was extracted. was produced in our study (Figure 1b). Another abundant The docked conformation graphics was generated using compound (labelled as C2 in Figure 1a and 1b) was eluted PyMOL 1.3 (Schrodinger, LLC) and the 2-D diagram was at a retention time of 11.70 min and detected in both sets computed by using Discovery Studio Client v4.5.0.15071 of enzymatic reactions. This compound was a by-product and LigPlot+ v1.4. (Laskowski and Swindells, 2011) for the of polyethyleneglycol (PEG), possibly derived from Triton hydrogen bonding, electrostatic, van der Waals, and pi X-100, after confirmation by LCMS (data not shown). interactions. 3.2. Binding site and homology model of CHS variant 2 The Ramachandran plot and PROCHECK results indicate 3. Results and discussion that the homology model is of good quality as the number 3.1. BrCHS var 2 substrates specificity of residues in the favored region was 92.9% (>90%) as The flavonoid biosynthesis pathway at KEGG pathway shown in supplementary material Figure B. As shown ko00941 (http://www.genome.jp/dbget-bin/www_ in Figure 2a, the multiple sequence alignment result of bget?ko00941) shows the formation of primary CHS CHS receptors from different plant species indicates that products, which are pinocembrin and naringenin. These the binding site of BrCHS var 2 receptor contains three CHS products are produced when the substrates used main amino acids, Cys 164, His 303, and Asn 336, that are cinnamoyl-CoA and p-coumaroyl-CoA, respectively form a catalytic triad. The catalytic triad is commonly (Mazumdar and Chattopadhyay, 2015). found in all type III polyketide synthase of Zingiberaceae The retention times for all compounds and substrates (Mallika et al., 2016). The surrounding active site residues identified in the HPLC chromatogram are listed in MMYQQGC164-AGGT and GFGPG loop are as shown in Table 1 and were referred prior to the CHS enzymatic Figure 2b.

Table 1. Retention time of four reference metabolites and substrates.

Reference compounds Retention time (min) Naringenin 6.531 ± 0.10 Naringenin chalcone 6.571 ± 0.10 Metabolites Pinocembrin 10.304 ± 0.10 Pinocembrin chalcone 8.913 ± 0.10 Malonyl-CoA 2.231 ± 0.10 Substrates Cinnamoyl-CoA 5.068 ± 0.10 p-Coumaroyl-CoA 3.256 ± 0.10

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a)

b)

Figure 1. a) HPLC chromatograms of CHS enzymatic activity assay in Set 1: malonyl-CoA with cinnamoyl-CoA of wild-type (top panel) and transformed cell suspension culture (bottom panel). b) HPLC chromatograms of CHS enzymatic activity assay in Set 2: malonyl- CoA with p-coumaroyl-CoA of wild-type (top panel) and transformed cell suspension culture (bottom panel). HPLC chromatogram detecting pinocembrin chalcone (C1) as shown in a) and nontargeted compound (naringenin and/or naringenin chalcone) detected in b). C2: Polyethyleneglycol (PEG).

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a)

Figure 2. a) Alignment of CHS deduced amino acid sequences from B. rotunda (BrCHS var 1-var 5), Cucurma longa (ClPKS9; JN017186.1), Curcuma alismatifolia (CaCHS; GU140082.1), Musa acuminata (MaPKSIII3; GU724609.1), Oryza sativa (OsCHS; AB000801.2), Zea mays (ZmCHS; NM_001155550.1), and Medicago sativa (MsCHS2; L02902.1). b) The amino acids of the catalytic Cys164-His303-Asn336 triad of BrCHS var 2 with active site residues MMYQQGC164-AGGT-H303-N336 (magenta) and GFGPG loop (yellow) are shown in cartoon (left) and surface (right) rendering.

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3.3. Molecular docking and docked conformation the substrate ligand, including the two gatekeeper residues analysis of CfCHS, Phe 266 and Phe 216 with π–π interactions. Based on the Table 2, cinnamoyl-CoA showed more Moreover, Lys 270 also formed hydrogen bonding with negative docked energy compared to p-coumaroyl- cinnamoyl-CoA (Awasthi et al., 2016). CoA. Thus, cinnamoyl-CoA has higher binding affinity The only structural difference between cinnamoyl- for BrCHS var 2 than p-coumaroyl-CoA. This result CoA and p-coumaroyl-CoA is the extra hydroxyl is also supported by several hydrogen bonds found in group at the para position on p-coumaroyl-CoA or cinnamoyl-CoA compared to p-coumaroyl-CoA. In the 4-hydroxycinnamoyl-CoA. As shown in Figures 3c and docking simulations, CoA and acetyl-CoA were used as 3d, cinnamoyl-CoA interacts with Cys 164 to initiate references. The binding of cinnamoyl-CoA to BrCHS var 2 the loading process of cinnamoyl moiety to Cys 164 as receptor was dominantly stabilized by hydrogen bonding thioester monoketide intermediate, which will proceed to and electrostatic interaction, whereas the binding of subsequent decarboxylation, elongation, and aromatization p-coumaroyl-CoA to BrCHS var 2 receptor was hindered (Ferrer et al., 1999) to produce pinocembrin. On the other by the repulsive electrostatic interaction with less hydrogen hand, the hydroxyl group of p-coumaroyl-CoA formed bonding and electrostatic interaction. Gln 161 (2.63), Leu an unfavorable interaction with Gln 161 and a hydrogen 258 (2.62), and Arg 259 (2.63) were the residues involved bond with Cys 164 (Figure 4d), which constitutes a steric in hydrogen bond formation with cinnamoyl-CoA. On hindrance to the subsequent formation of thioester the other hand, Cys 164 (3.13) was the only residue that monoketide intermediate and hence the final production interacted with p-coumaroyl-CoA through hydrogen of naringenin. Therefore, molecular docking and docked bonding. conformation analysis enables the elucidation of reaction Figures 3 and 4 show the docked conformations for mechanism of BrCHS var 2 in using cinnamoyl-CoA as cinnamoyl-CoA and p-coumaroyl-CoA, respectively. the preferred substrate instead of p-coumaroyl-CoA to The main interaction types were hydrogen bonding and produce pinocembrin. electrostatic with weaker interactions such as van der The formation of hydrogen bonding by Cys 164, His Waals, pi–pi, and pi–alkyl interactions. A similar result was 303, and Asn 336 with the thioester of malonyl-CoA reported by Awasthi et al. (2016) for CfCHS from Coleus is shown in Figure 5 and this will initiate subsequent forskohlii in which the preferred substrate was cinnamoyl- decarboxylation, carbanion formation, and further CoA compared to p-courmaroyl-CoA and it was found attack of cinnamoyl thioester on Cys 164 (Ferrer et al., that cinnamoyl-CoA showed a more negative docking 1999). The more subtle van der Waals interactions from score than p-coumaryol-CoA. They also indicated that Cys the gatekeeper residues Phe 215 and Phe 265 were also 164, His 304, and Asn 337 were identified to interact with observed in Figures 3–5. Supplementary material Figures

Table 2. Docked energy for ligands to BrCHS var 2 receptor.

Docked energy Electrostatic energy van der Waals energy Repulsive Ligand Hydrogen bond length (Å) (kcal/mol) (kcal/mol) (kcal/mol) interaction

Arg58 (2.55, 2.73, 3.35) CoA Lys62 (2.59, 2.62) –83.7 ± 5.8 –289.1 ± 4.1 –21.0 ± 1.3 0 (Reference) Cys164 (3.13) Asn336 (3.28)

Lys55 (2.62) Acetyl-CoA Lys62 (3.26) –63.5 ± 3.6 –212.2 ± 2.0 –18.1 ± 6.8 0 (Reference) Cys164 (3.13) His303 (2.76)

Lys62 (2.44, 2.56) Cys164 (3.16) Malonyl- CoA –62.7 ± 3.4 –223.9 ± 9.0 –18.0 ± 5.2 0 His303 (3.06) Asn336 (2.80)

Gln161 (2.63) Cinnamoyl- CoA –54.0 ± 3.0 –64.3 ± 14.7 –24.7 ± 5.8 Leu258 (2.62) 0 Arg259 (2.73) p-Coumaroyl- CoA –49.3 ± 4.3 –33.5 ± 14.0 –27.3 ± 1.8 Cys164 (3.13) 1

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c)

d)

Figure 3. Docked conformation of BrCHS var 2 with cinnamoyl-CoA rendering in (a) surface; (b) wireframe; (c) 2-D diagram; (d) interaction types. Cyan: CHS variant 2 receptor; yellow: amino acid around the binding site; CPK: cinnamoyl-CoA.

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c)

d)

Figure 4. Docked conformation of BrCHS var 2 with p-coumaroyl-CoA rendering in (a) surface; (b) wireframe; (c) 2-D diagram; (d) interaction types. Cyan: CHS variant 2 receptor; yellow: amino acid around the binding site; CPK: p-coumaroyl-CoA.

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Figure 5. 2-D diagram of docked conformation of BrCHS var 2 with malonyl-CoA.

C1 and C2 shows the 2-D interaction diagrams of acetyl- as a plant host and it would also pave the way for further CoA and CoA, respectively, to BrCHS var 2 as reference. in silico investigation of chalcone synthase and flavonoid In the present work, we found that the preferred biosynthesis by using molecular dynamics and shed light substrate for BrCHS var 2 was cinnamoyl-CoA to produce on its system biology pathway modelling. pinocembrin chalcone, which has been successfully verified by HPLC analysis. Docked confirmation analysis further Acknowledgments confirms the enzymatic assay results and we found that We are grateful for the University of Malaya’s research the binding of cinnamoyl-CoA to BrCHS var 2 receptor grants RP032-15AFR and RP032C-15AFR for supporting was with numerous hydrogen bonding and stronger this research work. We thank Dr Teh Ser Huy for providing electrostatic interaction with lower negative docked the amino acid sequences of the five variants of BrCHS. energy compared to p-coumaroyl-CoA. The findings of We also thank Ms Nurul Hana Mohd Yusof and Mr Md this investigation are useful for future in vitro production Arman Hossain for their assistance in the molecular of novel polyketides by utilizing cell suspension culture docking experiments.

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Supplementary material

Figure A. 1) In-house amino acid sequence for BrCHS var 2 protein; 2) Template sequence for homology modelling.

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PROCHECK Ramachandran Plot 1523016 - Chain A 180 B ~b b

135 b ~b ~l 90 l

45 L a A 0 ~a LYS 3 (A) Psi (degrees) -45

-90

SER 338 (A) -135 ~b ~p b p ~b

-180 -135 -90 -45 0 45 90 135 180 Phi (degrees) Plot statistics Residues in most favoured regions [A,B,L] 313 92.3% Residues in additional allowed regions [a,b,l,p] 24 7.1% Residues in generously allowed regions [~a,~b,~l,~p] 1 0.3% Residues in disallowed regions 1 0.3% ------Number of non-glycine and non-proline residues 339 100.0% Number of end-residues (excl. Gly and Pro) 2 Number of glycine residues (shown as triangles) 32 Number of proline residues 18 ---- Total number of residues 391

Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R-factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions.

1523016_01.ps Figure B. Ramachandran plot and PROCHECK result for BrCHS var 2 protein receptor homology model.

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Figure C. Docked conformation rendering in 2-D diagram for (C1) acetyl-CoA and (C2) CoA, respectively.

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