A 1.2 kb open reading frame of the PpCHS (Acession No: DQ 275627.2) was cloned in the BL21 (DE3) plysS using a PET32 (a) vector system. Specific primers [Forward primer (5′-CGGG CCA TTG AA ATG GCT TCT GCT-3′) and Reverse primer (5′-GAA TTC T CTA AGC GGA GTT GGGG-3′)] were designed to amplify the full-length of the open reading frame (ORF). The PpCHS was expressend as a thioredoxin (Trx)-fusion protein (~62 kDa) along with a Histidine-tag, which allows a single purification step if affinity chromatography using Ni²⁺ Sepharose resin is to be carried out. Thioredoxin is a 12-kD oxidoreductase enzyme containing a dithiol-disulfide active site, which may increase with the soluble, active, properly folded target protein by facilitating the formation of disulphide bonds in the cytoplasm. The purity of the purified PpCHS was analyzed through SDS-PAGE and NATIVE-PAGE as shown in Figure 1. The isoelectric point (pI) of PpCHS was determined to be approximately 7.1, however, the purified protein showed better migration under acidic conditions (pI 4.5) of NATIVE-PAGE. Apart from this, a molecular weight of approximately 120 kDa for PpCHS was determined by gel filtration (Sephacryl 200). The molecular weight of 62 kDa obtained on the SDS-PAGE indicates that the PpCHS is composed of two subunits.

Chalcone synthase catalyzes the formation of naringenin chalcone from p-coumaroyl-CoA and malonyl-CoA [1]. It catalyzes the condensation reaction of three acetyl units from malonyl- CoA with p-coumaroyl-CoA to produce naringenin chalcone [1]. However, recent reports have shown that PpCHS is capable of accepting hexanoyl-CoA as a starting molecule to produce hydroxyl-pyrone intermediates [10]. Reactions with different substrates such as hexanoyl-CoA were found to yield pyrone by-products, perhaps due to premature two condensation reactions between hexanoyl-CoA and malonyl-CoA [11]. Another type of polyketide synthase (PKS) from Cannabis sativa was also tested using hexanoyl-CoA as a substrate. It has been suggested that this enzyme converts one molecule of hexanoyl-CoA and three molecules of malonyl-CoA into olivetol. The proposed mechanism relates the formation of olivetol to Aldol-type cyclization [14]. Therefore, in this current work, hexanoyl-CoA was used as an alternative substrate to characterize and investigate the enzymatic properties of PpCHS.

Several factors, such as the time and the substrate concentration, were investigated. Based on the production of the cyclization products, only 37% relative activity was obtained using hexanoyl-CoA as a substrate relative to the p-coumaroyl-CoA (100%) catalyzed reaction [1]. Furthermore, due to the low conversion rates when using hexanoyl-CoA, the concentration of the substrates needed to maximize the production of the products was investigated.

Several different concentrations of hexanoyl-CoA (0.1 mM to 2.4 mM) were tested, as shown in Figure 2(a). These experiments revealed that 0.8 mM hexanoyl-CoA exhibited the highest production of products. No further increment was observed at higher substrate concentrations. In addition, the effect of the incubation time on the amount of product formed was studied. The results obtained revealed a significant increase after 30 min of incubation (Figure 2(b)). Likewise, the reaction of PKS from Cannabis sativa with hexanoyl-CoA showed no significant increase in the amount of product after 45 min of incubation [14].

Generally, chalcone synthase is known for its pH dependent activity. Therefore, the impact of these changes on the pyrone formation was determined. Interestingly, with hexanoyl-CoA as the substrate, the highest activity (in terms of pyrone formation) was found at pH 8, which is different from the optimum pH using p-coumaroyl-CoA, which is pH 7. Previous work has reported the formation of various chalcone analogs from butyryl-CoA and hexanoyl-CoA at pH 6.5, but no products were detected at pH 8.0 [12].

As is shown in Figure 3, at lower pHs, between 3 and 5, and at higher pHs, between 9 and 11, pyrone analogs are not produced, but other by-products were detected. Another polyketide synthase from Cannabis sativa produced olivetol from hexanoyl-CoA in a potassium phosphate buffer with pH 6.8 [14].

The recombinant PpCHS exhibited an optimum temperature of 30 °C (results not shown). A similar result was obtained for PKS from Cannabis sativa, for which the incubation reaction was perfomed at 30 °C [14]. A thermal stability study was performed by pre-incubating the chalcone synthase for 30 min at different temperatures in the range of 10 °C to 60 °C prior to the enzyme assay. According to the results obtained (Figure 4), the chalcone synthase activity increased at every 5 °C interval as the temperature increased up to 25 °C. At temperatures higher than 35 °C, there was a decrease in enzyme activity.

The three-dimensional structure of chalcone synthase from Medicago sativa shows the four main catalytic residues of the active site (Cys 164, His 303, Asn 336, Phe 215), conserved in other CHS-like enzymes [7]. In the crystal structure of CHS from Medicago sativa, the Cys 164 residue forms a hydrogen bond with the His 303 residue with an estimated distance of 3.5 Å. In the case of PpCHS, the predicted three-dimensional structure showed a calculated distance of 4 Å between the Cys 170 and His 309. Earlier, it had been proposed that the Cys 164 acted as a nucleophile, while His 303 acted as a general base by abstracting protons from Cys 164 to form a reactive thiolate for chalcone formation. Later on, through several mutational studies, the possibility of a stable imidazolium-thiolate ion pair formation between these two residues was suggested.

Apart from this, most mutational and chemical modification studies carried out to investigate the catalytic Cys-His interaction were often analyzed based on the production of naringenin chalcone using p-coumaroyl-CoA as a starting ester [15]. However, chalcone synthase’s unique characteristic is its ability to use various starter CoA esters. As previously reported, chalcone synthase from Physcomitrella patens exhibits 37% preference toward hexanoyl-CoA [1]. Therefore, this work describes the effect of Cys 170 Arg and Cys 170 Ser substitutions on the binding of other non-physiological starter CoA esters into the PpCHS’s catalytic cavity based on the production of pyrone derivatives.

Previously, it was found that PpCHS utilizes hexanoyl-CoA, producing significant amounts of polyketide products. In this study, HPLC analyses were carried out to quantify the new product formed. Based on the retention time of a 4-hydroxyl-6-methyl-2-pyrone standard as shown in Figure 5, the product was likely to be one of pyrone derivatives. Based on the results, both mutants were also able to utilize hexanoyl-CoA as a starter substrate, however, in this case, both mutants showed a lower production of pyrone product (Figure 6). Generally, the results showed that a single amino acid substitution, particularly the catalytic cysteine 170, affected the enzymatic pyrone production of PpCHS. These changes affecting the production of reaction products might be associated with the alteration in the binding capacity/affinity of the PpCHS catalytic site to the substrates. These results also agreed well with previous work done on chalcone synthase, which suggested that a decrease in or loss of the catalytic activity of the mutants might be caused by the effects of these substitutions on the covalent binding affinity of the starter molecule (hexanoyl-CoA) to the chalcone synthase active site cavity [16].

To further examine the validity of this data, homology modeling of PpCHS was performed to provide an insight into the effect of these mutations on the cavity volume of the catalytic site as well as the ion-pair interaction between the substituted residues with the His residue. The three-dimensional structure of PpCHS was built using the crystal structure of chalcone synthase from Medicago sativa,, which shares 63% sequence identity. Studies on the three-dimensional model structure of PpCHS revealed that the catalytic residues (Cys, His, Asn, Phe) of polyketide synthase are structurally conserved as illustrated in Figure 7. However, the active site cavity varies which leads to the formation of diverse products and by-products depending on the types of starter molecules used [7]. The calculated distance between the Cys 170 and His 309 as mentioned before is approximately 4 Å. The Cys-His interaction was found to be one of the most crucial points in the reaction mechanism of chalcone synthase.

It has been reported that the cavity volume plays an important role in the strength of the substrate binding interaction and hence any change in it might alter the product formation profile of the type III PKS. Cavity volume plays an important role in the strength of the substrate binding interaction. These cavities are important for molecular recognition to provide a direct connection to the exterior of the protein. The cavity volume of different type III PKS proteins, which includes chalcone synthase, stilbenecarboxylate synthase 2, stilbene synthase and 2-pyrone synthase, were calculated using CASTp, which is an algorithm based on computational geometry methods. It was found that the cavity volume of different type III PKS varied in a range of 737 Å–1683 Å [17].

In this study, the cavity volumes of the mutants (Cys 170 Arg, Cys 170 Ser) were measured using YASARA as listed in Table 1. Interestingly, despite low production of the reaction product by the mutants, the cavity volume for both mutants showed contradicting results in which the Cys 170 Arg mutant’s cavity volume decreased to 195.53 Å while the Cys 170 Ser mutant’s cavity volume increased to 231.23 Å. The substituted arginine residue, due to the presence of a guanidine group, tends to reside in the catalytic cavity of the active site, filling the space between the neighboring residues and leading to a smaller cavity volume of the mutant. However, due to the bulky structure of the guanidine group now occupying the space inside the active site, the positions of neighboring residues residing within the active site as well as the overall structure of the mutant are as shifted, leading to an increase in the Cys 170 Arg mutant’s structure volume to 43,391.02 Å. The increase of the volume of the structure reflects a more flexible and less compact structure which agrees well with the calculated total energy of Cys 170 Arg of −65.03 indicating a decrease of the mutant’s structure stability.

On the other hand, for the substituted serine residue, due to the presence of a smaller hydroxyl group, contributes to a larger space within the catalytic cavity of the active site which is 231.23 Å. Based on the YASARA structure prediction analysis, a hydrogen bond interaction was detected between the hydroxyl group of the Ser 170 and His 309. This interaction along with other possible interactions among other neighboring residues within the active site cavity might be one of the factors contributing to the decrease of the Ser 170 mutant structure volume in comparison with the Arg 170 mutant which is 43,330.84 Å. Therefore, the decrease in volume of the Ser 170 mutant reflects a structure compactness and stability of the whole structure which agrees well with the calculated total energy of −66.97.

This approach was also carried out on stilbene synthase, which evolved from CHS. The mutation of Cys to Ser showed a lower yield of product and an instability of the C164S mutant. Aside from this, other mutational studies carried out to mutate Cys at position 169 with Ser showed failure in detecting enzyme activity which defined the destruction of Cys as an essential role in the catalytic machinery of CHS [18]. However, in this work, some enzyme activity is still detectable by the mutant.

According to the thermal stability profile of recombinant chalcone synthase shown in Figure 4, a decrease in enzymatic activity was observed after pre-incubation (30 min) of enzyme at 35 °C prior to enzyme assay. Surprisingly, both Cys 170 Arg and Cys 170 Ser mutants showed increased enzymatic activity after pre-incubation of the mutant enzymes at 40 °C. Several case studies were done to investigate the effects of mutations on non-active site arginine residues of chalcone synthase. An arginine residue, largely protonated at physiological pH, plays an important role in forming salt bridges with other anionic amino acids. It was found that mutations on Arg 68, Arg 172, and Arg 328 greatly affected the enzyme activity due to their important roles in positioning the active site residues in correct catalytic topology. On the other hand, Arg 199 and Arg 350 were found to play an important role in maintaining the structural integrity and foldability of the enzyme [19].

In this study, the Cys 170 Arg mutant exhibited an increase in the thermal stability as is shown in Figure 8(a). Through the homology modeled structure of Cys 170 Arg mutant, there is a possible hydrogen-bonding interaction (1.744 Å) between the Arg 170 residue with Ile 260 located in the middle of a loop, maintaining the rigidity and stability of the whole protein structure (Figure 8(b)). Therefore, improvement in protein stability leads to an increase in the thermal stability of the mutants.

Cells harboring the recombinant plasmid were cultured in liquid LB medium (200 mL) containing 10 ug/mL ampicillin. When the optical density of the culture at 600 nm reached 0.5, isopropyl-β-d-thiogalactoside (1 mM) was added to the culture to induce recombinant protein expression. After incubation at 37 °C for 12 h, the cells were harvested by centrifugation, resuspended in 10 mL of buffer (20 mM phosphate, pH 7.4 containing 80 mM imidazole and 0.5 M NaCl), and lysed by sonication.

The homogenate was centrifuged at 12,000 rpm for 30 min to remove the insoluble material. Then, the supernatant was applied to a of Ni²⁺ Sepharose Fast Flow column equilibrated with binding buffer (20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl, 80 mM imidazole). Following a washing step with binding buffer (10 CV), the enzyme was eluted with 300 mM imidazole (20 CV).

Enzyme assays were performed according to [14] with modifications. The standard reaction mixture (500 μL) contained 25 μL of 0.8 mM malonyl-CoA, 25 μL of 0.4 mM hexanoyl-CoA, and 100 μL of enzyme in 20 mM phosphate buffer, pH 7 and the reactions were incubated at 30 °C for 2 h. After termination of the reaction with 1 N HCl, the reaction products were extracted for 2 min with ethyl acetate, and the upper layer was subjected to analysis using HPLC. The reaction products were identified with HPLC (Agilent) using a C18 Hypersil Gold column (5 μm, 4.6 mm × 150 mm) with a flow rate of 0.5 mL/min and using a 280 nm UV detector. The chromatographic separation was performed using a gradient of acetonitrile (30–60% in 30 min) in water.

The C170R and C170S mutants were generated using the QuickChange site-directed mutagenesis kit (Stratagene, USA). Specific mutagenic primers were design to amplify the open reading frame of PpCHS gene.

C170S mutant: (Forward primer 5′-ATG ATG TAC CAA ACC GGG TCT TTC GGC GGT GCA TCC GTG-3′) and (Reverse primer 5′-CAC GGA TGC ACC GCC GAA AGA CCC GGT TTG GTA CAT CAT-3′).

C170R mutant: (Forward primer 5′-ATG ATG TAC CAA ACC GGG AGG TTC GGC GGT GCA TCC GTG-3′) and (Reverse primer 5′-CAC GGA TGC ACC GCC GAA CCT CCC GGT TTG GTA CAT CAT-3′).

After confirmation of the sequences, the plasmids that expressed the mutants were transformed in E. coli BL21 (DE3) pLysS respectively.

Both recombinant PpCHS and C170S and C170R mutants were grown to A600~0.5, and induced with 1 mm IPTG for 12 h incubation time. The cells were harvested at 8,000 rpm for 10 min at 4 °C. The pellet was suspended with phosphate buffer prior to sonication and the supernatant collected after centrifugation at 12,000 rpm was used for enzyme activity determination.

For HPLC analysis, the reaction products were identified using a Nucleosil C18 column with a flow rate of 0.1 mL/min at UV 280 nm detection. The chromatography was run using a gradient of Acetonitrile (30–70%) in water. Under these conditions, 4-hydroxyl-6-methyl-2-pyrone and naringenin were used as an internal standard. The authentic standard of 1 mg/mL of 4-hydroxy-6-metyl-2-pyrone was eluted at a retention time of 1.65 min which is comparable with the formation of mutant’s reaction products. For liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection of unknown compounds, the positive ionization mode was chosen as the detection mode with the collision gas off and was set at 370 °C. The positive ion MRM chromatograms of chalcone synthase reaction product at m/z 272.50–273.50 were obtained from chalcone synthase reaction product containing 100 ng/mL naringenin which further confirms the formation of a chalcone analoque from the reaction products [10].

From the output of PSI-BLAST and BLAST in search of a suitable template with a PDB structure, chalcone synthase from Medicago sativa with PDB ID: IBQ6 was selected with 63% sequence identity. It is important to obtain the best sequence alignment between the template protein and the model sequence. Therefore, multiple sequence alignments of target sequences with the selected template sequences were performed using structural alignments to establish a baseline for the accuracy of the sequence alignments. Finally, the homology model of Physcomitrella patens chalcone synthase (Accession No: DQ 275627.2) was constructed using the Accelerys Discovery Studio suite program.

MODELER Accelrys, San Diego [20] allows the autonomic generation of one or more hypothetical models based upon one or more templates [21]. Once a target-template alignment was constructed, MODELER automatically calculated a 3D model of the target completely. This method generated a PpCHS model structure based on the IBQ6 (Medicago sativa chalcone synthase).