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![SDS/PAGE (A) and radio-TLC (B, C) of the wild-type and mutant chalcone and stilbene synthases (A) Soluble (s) and insoluble (i) E. coli extracts and purified (p) Trx-CHS and Trx-STS enzymes (60 kDa, arrow) are shown. Proteins were separated on 12 % (w/v) polyacrylamide mini-slab gels under reducing conditions, followed by staining with Coomassie Brilliant Blue R250. Lane M, molecular mass markers (molecular masses are shown in kDa at the right). (B, C) Radio-TLC of CHS and STS reaction products. Condensing activities of wild-type and P 375 G mutants of CHS and STS (B) were determined with p-coumaroyl-CoA and [2-14 C]malonyl-CoA as substrates. The cyclization products (naringenin and resveratrol) as well as derailment products (BNY and CTAL) were separated and quantified by using reversed-phase (C 18 ) TLC and an Imaging Plate Analyzer (BAS2000 ; Fuji). The products were identified by using authentic samples or by a comparison of their known R F values [15]. C. Malonyl-CoA decarboxylation (acetyl-CoA formation) activity (C) was assayed with [2-14 C]malonyl-CoA as a sole substrate. Acetyl-CoA (a) and malonyl-CoA (m) were separated on a silica 60 TLC plate and identified by comparing their R F values [4]. The reactions were performed with 1 µg of the wild-type enzyme, 5 µg of the P 375 G mutant enzyme or 15 µg of the G 374 L mutant enzyme. o, TLC origin ; f, solvent front.](profile/Dae-Yeon-Suh/publication/12393323/figure/fig2/AS:601681574244391@1520463474876/SDS-PAGE-A-and-radio-TLC-B-C-of-the-wild-type-and-mutant-chalcone-and-stilbene.png)
SDS/PAGE (A) and radio-TLC (B, C) of the wild-type and mutant chalcone and stilbene synthases (A) Soluble (s) and insoluble (i) E. coli extracts and purified (p) Trx-CHS and Trx-STS enzymes (60 kDa, arrow) are shown. Proteins were separated on 12 % (w/v) polyacrylamide mini-slab gels under reducing conditions, followed by staining with Coomassie Brilliant Blue R250. Lane M, molecular mass markers (molecular masses are shown in kDa at the right). (B, C) Radio-TLC of CHS and STS reaction products. Condensing activities of wild-type and P 375 G mutants of CHS and STS (B) were determined with p-coumaroyl-CoA and [2-14 C]malonyl-CoA as substrates. The cyclization products (naringenin and resveratrol) as well as derailment products (BNY and CTAL) were separated and quantified by using reversed-phase (C 18 ) TLC and an Imaging Plate Analyzer (BAS2000 ; Fuji). The products were identified by using authentic samples or by a comparison of their known R F values [15]. C. Malonyl-CoA decarboxylation (acetyl-CoA formation) activity (C) was assayed with [2-14 C]malonyl-CoA as a sole substrate. Acetyl-CoA (a) and malonyl-CoA (m) were separated on a silica 60 TLC plate and identified by comparing their R F values [4]. The reactions were performed with 1 µg of the wild-type enzyme, 5 µg of the P 375 G mutant enzyme or 15 µg of the G 374 L mutant enzyme. o, TLC origin ; f, solvent front.
Source publication
Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing e...
Contexts in source publication
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... enzymes were expressed as Trx fusion proteins and they were joined with the Trx protein at their N-ends via a linker containing the His ' tag and an enterokinase cleavage site. These Trx fusion proteins are hereafter referred to as CHS and STS unless otherwise noted. The His ' tag enabled us to obtain the wild-type enzyme and the P$(&G mutants in apparent homogeneity after a single purification step with Ni# + -chelation chromatography ( Figure 3A). Mutant enzymes were purified under conditions that were essentially identical with those used for the wild-type enzymes. The Pro-375 Gly mutation in both CHS and STS apparently disturbed the folding process so that most of the expressed P$(&G mutants were recovered in an insoluble fraction as inclusion bodies. This resulted in an approx. 4-fold decrease in the yields of the purified mutants in comparison with the wild- type enzymes (Table 1). The Gly-374 Leu mutation exhibited more adverse effects during expression, especially in the STS- G$(%L mutant. A time-course study nevertheless revealed a peak in expression level at 10-12 h after induction as judged by SDS\PAGE (results not shown). Hence both the G$(%L mutants were harvested after 10 h induction at 25 mC. Owing to lower expression levels and solubilities, the G$(%L mutants were only partly purified by Ni# + -chelation chromatography ( Figure ...
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... enzymes were expressed as Trx fusion proteins and they were joined with the Trx protein at their N-ends via a linker containing the His ' tag and an enterokinase cleavage site. These Trx fusion proteins are hereafter referred to as CHS and STS unless otherwise noted. The His ' tag enabled us to obtain the wild-type enzyme and the P$(&G mutants in apparent homogeneity after a single purification step with Ni# + -chelation chromatography ( Figure 3A). Mutant enzymes were purified under conditions that were essentially identical with those used for the wild-type enzymes. The Pro-375 Gly mutation in both CHS and STS apparently disturbed the folding process so that most of the expressed P$(&G mutants were recovered in an insoluble fraction as inclusion bodies. This resulted in an approx. 4-fold decrease in the yields of the purified mutants in comparison with the wild- type enzymes (Table 1). The Gly-374 Leu mutation exhibited more adverse effects during expression, especially in the STS- G$(%L mutant. A time-course study nevertheless revealed a peak in expression level at 10-12 h after induction as judged by SDS\PAGE (results not shown). Hence both the G$(%L mutants were harvested after 10 h induction at 25 mC. Owing to lower expression levels and solubilities, the G$(%L mutants were only partly purified by Ni# + -chelation chromatography ( Figure ...
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... the CHS and STS reactions in itro, BNY and CTAL are also produced as derailment products by hydrolysis of the growing chain at the stage of triketide and tetraketide respectively (Figures 1 and 3B) [6,15,18]. The detection method (acidification of reaction mixture followed by extraction and RP-TLC analysis) was chosen to follow both the production of not only the final products but also the derailment products so as to assess the mutational effects on the active site. CHS and STS also catalyse malonyl-CoA decarboxylation and CO # exchange in both the presence and the absence of p-coumaroyl-CoA [19,20]. The decarboxylation of malonyl-CoA initially produces an acetyl- CoA carbanion, which is subsequently protonated to acetyl-CoA (Figure 1). In the present study, acetyl-CoA formation was followed, to measure malonyl-CoA decarboxylation activity in the absence of p-coumaroyl-CoA ( Figure ...
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... K m (app) and V max values for the wild-type STS and STS- P$(&G mutant were not significantly different, indicating that the Pro-375 Gly mutation did not significantly affect the malonyl- CoA binding and decarboxylation reaction of STS. However, the same mutation had a drastic effect on the product profile. Whereas the product profile in the wild-type STS reaction was resveratrol CTAL $ BNY naringenin, it changed to CTAL resveratrol$ BNY naringenin in the STS-P$(&G reaction (Table 1 and Figure 3B). Clearly, the ability of this mutant to catalyse the STS-type cyclization to produce resveratrol was greatly impaired. The concurrent increase in production of BNY and CTAL suggested that, in contrast with CHS-P$(&G, ' loosening up ' of the active site in STS-P$(&G had a Table 1 Expression, product profile and enzyme activity of the wild-type and mutant chalcone and stilbene synthases Results are meanspS.D. (n l 3-6). Yields are those of purified enzymes obtained after a Ni 2 + -chelation chromatography step. For determining the product profiles and measuring the specific activity of the condensing reaction, the reaction was performed in 0.1 M potassium phosphate, pH 7.2, containing 10 % (v/v) glycerol, 0.1 % (v/v) Triton X-100 and 1 mM dithiothreitol with 0.1 mM p-coumaroyl-CoA and 16.8 µM [2-14 C]malonyl-CoA as substrates. Specific activity is defined as pmol of C 2 units condensed/s per mg of protein (pkat/mg). For measuring the specific activity of the malonyl-CoA decarboxylation, the reaction was performed in 0.1 M Hepes, pH 7.0, with 100 µM [2-14 C]malonyl-CoA as a sole substrate. The amounts of enzymes added to the reaction were : wild-type enzymes, 1 µg ; P375G mutants, 5 µg ; G374L mutants, 15 µg. Specific activity is defined as pmol of acetyl-CoA produced/s per mg of protein (pkat/mg). ' Inactive ' signifies that even with 30 µg of partly purified mutant enzyme (Figure 3), the amounts of any products were below the detection limit. major role. Further, STS-P$(&G exhibited an increased cross- reaction [15] of up to 30 %. That is, the mutant catalysed almost one CHS-type cyclization for every three STS-type cyclization reactions. It should be noted that the increased cross-reaction cannot be attributed solely to the decreased production of resveratrol because the specific activity for naringenin production by STS-P$(&G (1.6p0.60 pkat\mg, meanpS.D.) remained simi- lar to that of the wild-type enzyme (1.6p0.41 pkat\mg) in spite of the 4.7-fold decrease in condensing ability and the 4.4-fold decrease in malonyl-CoA decarboxylation activity (Table 1). This unexpected result could best be explained by the fact that different cyclization reactions in CHS and STS are indeed controlled by differences in the active-site geometry, which determines the way in which the linear intermediate folds before cyclization [14,21]. It is therefore proposed that the active sites of CHS and STS are configured to favour one type of spatial folding of the intermediate. The P$(&G mutation alters the STS active site to allow both types of folding to occur partly by ' loosening up ' (see the Discussion section). As a way of assessing whether the mutant retained the conformational integrity of the wild-type enzyme, kinetic par- ameters of the wild-type and mutant enzymes were compared. As summarized in Table 2, the Pro-375 Gly mutation in CHS and STS had a limited effect on the K m (app) values for both substrates, suggesting that the mutants retained the wild-type tertiary structures. The identity of the products formed by STS- P$(&G was confirmed by LC-APCIMS analysis in positive-ion mode. The results were in complete agreement with earlier reports [6,15] No condensing activity was detected with the G$(%L mutants of CHS and STS, even after larger amounts of proteins were added to the reaction. Some residual malonyl-CoA activities were detected but they were less than 1 % of the corresponding wild-type activities. Because of the lower expression and solubility of the G$(%L mutants, the possibility cannot be excluded that the almost complete loss of the enzyme activity is due to minimal amounts of functionally folded enzymes. However, considering the fact that partly purified preparations contained the mutants as the major protein ( Figure 3A), it seems more likely that the loss of activity was due either to the blocking of active sites by the bulky side chain of Leu or to the impaired flexibility of the ...
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... K m (app) and V max values for the wild-type STS and STS- P$(&G mutant were not significantly different, indicating that the Pro-375 Gly mutation did not significantly affect the malonyl- CoA binding and decarboxylation reaction of STS. However, the same mutation had a drastic effect on the product profile. Whereas the product profile in the wild-type STS reaction was resveratrol CTAL $ BNY naringenin, it changed to CTAL resveratrol$ BNY naringenin in the STS-P$(&G reaction (Table 1 and Figure 3B). Clearly, the ability of this mutant to catalyse the STS-type cyclization to produce resveratrol was greatly impaired. The concurrent increase in production of BNY and CTAL suggested that, in contrast with CHS-P$(&G, ' loosening up ' of the active site in STS-P$(&G had a Table 1 Expression, product profile and enzyme activity of the wild-type and mutant chalcone and stilbene synthases Results are meanspS.D. (n l 3-6). Yields are those of purified enzymes obtained after a Ni 2 + -chelation chromatography step. For determining the product profiles and measuring the specific activity of the condensing reaction, the reaction was performed in 0.1 M potassium phosphate, pH 7.2, containing 10 % (v/v) glycerol, 0.1 % (v/v) Triton X-100 and 1 mM dithiothreitol with 0.1 mM p-coumaroyl-CoA and 16.8 µM [2-14 C]malonyl-CoA as substrates. Specific activity is defined as pmol of C 2 units condensed/s per mg of protein (pkat/mg). For measuring the specific activity of the malonyl-CoA decarboxylation, the reaction was performed in 0.1 M Hepes, pH 7.0, with 100 µM [2-14 C]malonyl-CoA as a sole substrate. The amounts of enzymes added to the reaction were : wild-type enzymes, 1 µg ; P375G mutants, 5 µg ; G374L mutants, 15 µg. Specific activity is defined as pmol of acetyl-CoA produced/s per mg of protein (pkat/mg). ' Inactive ' signifies that even with 30 µg of partly purified mutant enzyme (Figure 3), the amounts of any products were below the detection limit. major role. Further, STS-P$(&G exhibited an increased cross- reaction [15] of up to 30 %. That is, the mutant catalysed almost one CHS-type cyclization for every three STS-type cyclization reactions. It should be noted that the increased cross-reaction cannot be attributed solely to the decreased production of resveratrol because the specific activity for naringenin production by STS-P$(&G (1.6p0.60 pkat\mg, meanpS.D.) remained simi- lar to that of the wild-type enzyme (1.6p0.41 pkat\mg) in spite of the 4.7-fold decrease in condensing ability and the 4.4-fold decrease in malonyl-CoA decarboxylation activity (Table 1). This unexpected result could best be explained by the fact that different cyclization reactions in CHS and STS are indeed controlled by differences in the active-site geometry, which determines the way in which the linear intermediate folds before cyclization [14,21]. It is therefore proposed that the active sites of CHS and STS are configured to favour one type of spatial folding of the intermediate. The P$(&G mutation alters the STS active site to allow both types of folding to occur partly by ' loosening up ' (see the Discussion section). As a way of assessing whether the mutant retained the conformational integrity of the wild-type enzyme, kinetic par- ameters of the wild-type and mutant enzymes were compared. As summarized in Table 2, the Pro-375 Gly mutation in CHS and STS had a limited effect on the K m (app) values for both substrates, suggesting that the mutants retained the wild-type tertiary structures. The identity of the products formed by STS- P$(&G was confirmed by LC-APCIMS analysis in positive-ion mode. The results were in complete agreement with earlier reports [6,15] No condensing activity was detected with the G$(%L mutants of CHS and STS, even after larger amounts of proteins were added to the reaction. Some residual malonyl-CoA activities were detected but they were less than 1 % of the corresponding wild-type activities. Because of the lower expression and solubility of the G$(%L mutants, the possibility cannot be excluded that the almost complete loss of the enzyme activity is due to minimal amounts of functionally folded enzymes. However, considering the fact that partly purified preparations contained the mutants as the major protein ( Figure 3A), it seems more likely that the loss of activity was due either to the blocking of active sites by the bulky side chain of Leu or to the impaired flexibility of the ...
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... K m (app) and V max values for the wild-type STS and STS- P$(&G mutant were not significantly different, indicating that the Pro-375 Gly mutation did not significantly affect the malonyl- CoA binding and decarboxylation reaction of STS. However, the same mutation had a drastic effect on the product profile. Whereas the product profile in the wild-type STS reaction was resveratrol CTAL $ BNY naringenin, it changed to CTAL resveratrol$ BNY naringenin in the STS-P$(&G reaction (Table 1 and Figure 3B). Clearly, the ability of this mutant to catalyse the STS-type cyclization to produce resveratrol was greatly impaired. The concurrent increase in production of BNY and CTAL suggested that, in contrast with CHS-P$(&G, ' loosening up ' of the active site in STS-P$(&G had a Table 1 Expression, product profile and enzyme activity of the wild-type and mutant chalcone and stilbene synthases Results are meanspS.D. (n l 3-6). Yields are those of purified enzymes obtained after a Ni 2 + -chelation chromatography step. For determining the product profiles and measuring the specific activity of the condensing reaction, the reaction was performed in 0.1 M potassium phosphate, pH 7.2, containing 10 % (v/v) glycerol, 0.1 % (v/v) Triton X-100 and 1 mM dithiothreitol with 0.1 mM p-coumaroyl-CoA and 16.8 µM [2-14 C]malonyl-CoA as substrates. Specific activity is defined as pmol of C 2 units condensed/s per mg of protein (pkat/mg). For measuring the specific activity of the malonyl-CoA decarboxylation, the reaction was performed in 0.1 M Hepes, pH 7.0, with 100 µM [2-14 C]malonyl-CoA as a sole substrate. The amounts of enzymes added to the reaction were : wild-type enzymes, 1 µg ; P375G mutants, 5 µg ; G374L mutants, 15 µg. Specific activity is defined as pmol of acetyl-CoA produced/s per mg of protein (pkat/mg). ' Inactive ' signifies that even with 30 µg of partly purified mutant enzyme (Figure 3), the amounts of any products were below the detection limit. major role. Further, STS-P$(&G exhibited an increased cross- reaction [15] of up to 30 %. That is, the mutant catalysed almost one CHS-type cyclization for every three STS-type cyclization reactions. It should be noted that the increased cross-reaction cannot be attributed solely to the decreased production of resveratrol because the specific activity for naringenin production by STS-P$(&G (1.6p0.60 pkat\mg, meanpS.D.) remained simi- lar to that of the wild-type enzyme (1.6p0.41 pkat\mg) in spite of the 4.7-fold decrease in condensing ability and the 4.4-fold decrease in malonyl-CoA decarboxylation activity (Table 1). This unexpected result could best be explained by the fact that different cyclization reactions in CHS and STS are indeed controlled by differences in the active-site geometry, which determines the way in which the linear intermediate folds before cyclization [14,21]. It is therefore proposed that the active sites of CHS and STS are configured to favour one type of spatial folding of the intermediate. The P$(&G mutation alters the STS active site to allow both types of folding to occur partly by ' loosening up ' (see the Discussion section). As a way of assessing whether the mutant retained the conformational integrity of the wild-type enzyme, kinetic par- ameters of the wild-type and mutant enzymes were compared. As summarized in Table 2, the Pro-375 Gly mutation in CHS and STS had a limited effect on the K m (app) values for both substrates, suggesting that the mutants retained the wild-type tertiary structures. The identity of the products formed by STS- P$(&G was confirmed by LC-APCIMS analysis in positive-ion mode. The results were in complete agreement with earlier reports [6,15] No condensing activity was detected with the G$(%L mutants of CHS and STS, even after larger amounts of proteins were added to the reaction. Some residual malonyl-CoA activities were detected but they were less than 1 % of the corresponding wild-type activities. Because of the lower expression and solubility of the G$(%L mutants, the possibility cannot be excluded that the almost complete loss of the enzyme activity is due to minimal amounts of functionally folded enzymes. However, considering the fact that partly purified preparations contained the mutants as the major protein ( Figure 3A), it seems more likely that the loss of activity was due either to the blocking of active sites by the bulky side chain of Leu or to the impaired flexibility of the ...
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... Pro-375 was mutated to Gly in CHS, specific activities calculated for the condensation reaction (total C # units condensed for all the products\s per mg of protein) and malonyl-CoA decarboxylation decreased 5.9-fold and 6.8-fold respectively (Table 1). However, a smaller decrease in V max values for condensation (3.1-fold) and for decarboxylation (1.6-fold) in- dicated that Pro-375 has a limited role in the early step of malonyl-CoA decarboxylation (Table 2) and that the larger differences in specific activities were due partly to different degrees of purity. More interestingly, the product profile of CHS-P$(&G was also changed. Whereas the ratio of CTAL to naringenin produced by the wild-type CHS was less than 0.4, the same ratio in the CHS-P$(&G-catalysed reaction increased to nearly 0.9 under identical reaction conditions ( Figure 3B). In contrast, BNY production remained relatively constant (5-7 % of total condensations) in the wild-type CHS and CHS-P$(&G. These results indicated that selectively increased CTAL pro- duction by the P$(&G mutant was not simply due to a ' loosening up ' of the active site caused by increased flexibility in the loop. This ' loosening up ' effect would have resulted in an increased access of solvent water to the growing intermediates, leading to an increased production of both derailment products. Rather, it seemed more likely that the change in the active-site configuration of the CHS-P$(&G mutant impeded proper CHS-type folding of the enzyme-bound tetraketide intermediate, which should even- tually be hydrolysed by solvent water. Once hydrolysed, the resulting free tetraketide could no longer undergo the CHS-type cyclization because the cyclization (intramolecular Claisen con- densation) requires an activated carbonyl carbon (Figure 1). As a result, the free tetraketide probably diffuses out of the active site and undergoes spontaneous lactonization to give ...
Citations
... Sequence analysis showed that the predicted protein sequence of AaCHS bore all the hallmarks of functional CHS families ( Figure 3a). These included the presence of a strictly conserved catalytic site consisting of four residues: Cys164, His303, Asn336, and Phe215, corresponding to the crystal structure of Medicago sativa MsCHS2 [41], and a characteristic CHS signature, GFGPG, which modulates stereochemistry processes in the cyclization reaction [42]. Phylogenetic analysis (Figure 3b) showed the predicted AaCHS was closely related to the Morus notabilis homolog, MnCHS2 ...
... Sequence analysis showed that the predicted protein sequence of AaCHS bore all the hallmarks of functional CHS families (Figure 3a). These included the presence of a strictly conserved catalytic site consisting of four residues: Cys164, His303, Asn336, and Phe215, corresponding to the crystal structure of Medicago sativa MsCHS2 [41], and a characteristic CHS signature, GFGPG, which modulates stereochemistry processes in the cyclization reaction [42]. Phylogenetic analysis (Figure 3b) showed the predicted AaCHS was closely related to the Morus notabilis homolog, MnCHS2 (Genbank acc. ...
Breadfruit (Artocarpus altilis) is a traditional fruit tree of 15–30 m height in the tropics. The presence of size-controlling rootstock in the species is not known. A small tropical tree species, lakoocha (Artocarpus lakoocha), was recently identified as a potential vigor-controlling rootstock, conferring over a 65% reduction in breadfruit tree height. To better understand the intriguing scion/rootstock interactions involved in dwarfing, we investigate flavonoid accumulation and its regulation in breadfruit scions in response to different rootstocks. To this end, we isolated a chalcone synthase cDNA, AaCHS, and a full-length bifunctional dihydroflavonol 4-reductase cDNA, AaDFR, from breadfruit scion stems. The expression of both AaCHS and AaDFR genes was examined over the period of 16 to 24 months following grafting. During the development of the dwarf phenotype, breadfruit scion stems on lakoocha rootstocks display significant increases in total flavonoid content, and show upregulated AaCHS expression when compared with those on self-grafts and non-grafts. There is a strong, positive correlation between the transcript levels of AaCHS and total flavonoid content in scion stems. The transcript levels of AaDFR are not significantly different across scions on different rootstocks. This work provides insights into the significance of flavonoid biosynthesis in rootstock-induced breadfruit dwarfing.
... chalcone synthase) are involved in the biosynthesis of diverse secondary metabolites (e.g. flavonoids) and possess the catalytic Cys-His-Asn triad and the signature sequence, (A/G) FGPG (Suh et al., 2000;Abe & Morita, 2010). Additional ASCLs have been found by performing BLAST searches to interrogate genomes of interest and screening protein models followed by phylogenetic analysis. ...
... The HPLC results indicated that the catalytic activity of DsBBS was specific, using 4-coumaroyl-CoA as the substrate for resveratrol production. The Vmax of DsBBS-HisTag protein was 0.88 pmol s −1 mg −1 for resveratrol generation, which was slightly higher than 0.68 pmol s −1 mg −1 of PpCHS in Physcomitrella patens [26] and 0.46 pmol s −1 mg −1 of PlCHS in Pueraria lobata [27]. ...
Dendrobium sinense, an endemic medicinal herb in Hainan Island, is rich in bibenzyls. However, the key rate-limited enzyme involved in bibenzyl biosynthesis has yet to be identified in D. sinense. In this study, to explore whether there is a significant difference between the D. sinense tissues, the total contents of bibenzyls were determined in roots, pseudobulbs, and leaves. The results indicated that roots had higher bibenzyl content than pseudobulbs and leaves. Subsequently, transcriptomic sequencings were conducted to excavate the genes encoding type III polyketide synthase (PKS). A total of six D. sinense PKS (DsPKS) genes were identified according to gene function annotation. Phylogenetic analysis classified the type III DsPKS genes into three groups. Importantly, the c93636.graph_c0 was clustered into bibenzyl synthase (BBS) group, named as D. sinense BBS (DsBBS). The expression analysis by FPKM and RT-qPCR indicated that DsBBS showed the highest expression levels in roots, displaying a positive correlation with bibenzyl contents in different tissues. Thus, the recombinant DsBBS-HisTag protein was constructed and expressed to study its catalytic activity. The molecular weight of the recombinant protein was verified to be approximately 45 kDa. Enzyme activity analysis indicated that the recombinant DsBBS-HisTag protein could use 4-coumaryol-CoA and malonyl-CoA as substrates for resveratrol production in vitro. The Vmax of the recombinant protein for the resveratrol production was 0.88 ± 0.07 pmol s−1 mg−1. These results improve our understanding with respect to the process of bibenzyl biosynthesis in D. sinense.
... Cys-His-Asn triad and signature sequence (G/A)FGPG (Suh et al. 2000). Type III PKSs from Marchantia polymorpha, Ceratodon purpureus and Sphagnum fallax were identified by tBLASTn searches against their genome sequences in Phytozome 13 (phytozome-next.jgi.doe.gov) with PpORS as query sequence. ...
Main conclusion
PpORS-produced 2′-oxo-5-pentacosylresorcinol (2′-oxo-C25-RL) restored dehydration tolerance in ors-3, a knockout mutant of PpORS. Feeding experiments with [¹⁴C]-2′-oxo-C25-RL suggested the role of PpORS products in cuticular polymer that confer dehydration resistance.
Abstract
2′-Oxoalkylresorcinol synthase from the moss Physcomitrium (Physcomitrella) patens (PpORS) is the earliest diverged member of plant type III polyketide synthases, and produces very-long-chain 2′-oxoalkylresorcinols in vitro. Targeted knockouts of PpORS (ors) exhibited an abnormal phenotype (increased susceptibility to dehydration), and a defective cuticle in ors was suggested (Li et al., Planta 247:527–541, 2018). In the present study, we investigated chemical rescue of the ors phenotype and also metabolic fates of the PpORS products in the moss. Using C24-CoA as substrate, 2′-oxo-5-pentacosylresorcinol (2′-oxo-C25-RL) and two minor pyrones were first enzymatically prepared as total in vitro products. When a knockout mutant (ors-3) and control strains were grown in the presence of the total in vitro products or purified 2′-oxo-C25-RL, the ability of ors-3 and the control to survive dehydration stress increased in a dose-dependent manner. Structurally analogous long-chain alkylresorcinols also rescued the ors phenotype, although less efficiently. When the moss was grown in the presence of ¹⁴C-radiolabeled 2′-oxo-C25-RL, 96% of the radioactivity was recovered only after acid hydrolysis. These findings led us to propose that 2′-oxoalkylresorcinols are the functional in planta products of PpORS and are incorporated into cuticular biopolymers that confer resistance to dehydration. In addition, the earliest diverging ORS clade in phylogenetic trees of plant type III PKSs exclusively comprises bryophyte enzymes that share similar active site substitutions with PpORS. Further studies on these bryophyte enzymes may shed light on their roles in early plant evolution and offer a novel strategy for improving dehydration tolerance in plants.
... For instance, OsCHS2, OsCHS11, OsCHS12, OsCHS24 and OsCHS26 included a tyrosine (Y265), OsCHS9, OsCHS13, OsCHS14 and OsCHS15 contained glycine (G265), OsCHS3 and OsCHS28 included an isoleucine (I265), and OsCHS25 contained a cysteine (C265) as a substitute for F265, which probably caused their functional diversity and divergent enzymatic activities of OsCHS genes. In addition, as a CHS/STS characteristic signature sequence [107], the WGVLFGFGP375GLT motif was also maintained in almost all 28 OsCHS genes (Supplementary Figure S1), although their conserved motif mentioned above might show some substituted residues in some cases. Plant CHSs further fell into three distinct clades based on the phylogenetic relationships of OsCHSs and their homologs from other plant species on the NCBI (Supplementary Figure S2 and Supplementary Table S1). ...
Flavonoids are a class of key polyphenolic secondary metabolites with broad functions in plants, including stress defense, growth, development and reproduction. Oryza sativa L. (rice) is a well-known model plant for monocots, with a wide range of flavonoids, but the key flavonoid biosynthesis-related genes and their molecular features in rice have not been comprehensively and systematically characterized. Here, we identified 85 key structural gene candidates associated with flavonoid biosynthesis in the rice genome. They belong to 13 families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin synthase (ANS), flavone synthase II (FNSII), flavanone 2-hydroxylase (F2H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). Through structural features, motif analyses and phylogenetic relationships, these gene families were further grouped into five distinct lineages and were examined for conservation and divergence. Subsequently, 22 duplication events were identified out of a total of 85 genes, among which seven pairs were derived from segmental duplication events and 15 pairs were from tandem duplications, demonstrating that segmental and tandem duplication events play important roles in the expansion of key flavonoid biosynthesis-related genes in rice. Furthermore, these 85 genes showed spatial and temporal regulation in a tissue-specific manner and differentially responded to abiotic stress (including six hormones and cold and salt treatments). RNA-Seq, microarray analysis and qRT-PCR indicated that these genes might be involved in abiotic stress response, plant growth and development. Our results provide a valuable basis for further functional analysis of the genes involved in the flavonoid biosynthesis pathway in rice.
... Amino acid sequence comparison of A. thaliana AtCHS and SmCHSs to Medicago sativa MsCHS2 that has crystal structure available [6], AtCHS and SmCHSs showed that all CHSs contained the catalytic triad Cys164-His303-Asn336 (hereafter residue numbers refer to MsCHS2) and the gatekeeper Phe215 (Supplementary Figure S3). The G372FGPG residue, a CHS signature sequence that provides stereo-control during the cyclization [26], exists in MsCHS2, AtCHS and six SmCHSs including SmCHS1, SmCHS3-SmCHS5, SmCHS7, and SmCHS8. In addition, MsCHS2, AtCHS and SmCHS1 contain Thr197, Gly256 and Ser338, three residues shaping the 4-coumaroly-CoA binding pocket and the polyketide cyclization pocket (Supplementary Figure S3). ...
Flavonoids are a class of important secondary metabolites with a broad spectrum of pharmacological functions. Salviamiltiorrhiza Bunge (Danshen) is a well-known traditional Chinese medicinal herb with a broad diversity of flavonoids. However, flavonoid biosynthetic enzyme genes have not been systematically and comprehensively analyzed in S.miltiorrhiza. Through genome-wide prediction and molecular cloning, twenty six flavonoid biosynthesis-related gene candidates were identified, of which twenty are novel. They belong to nine families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), respectively. Analysis of intron/exon structures, features of deduced proteins and phylogenetic relationships revealed the conservation and divergence of S.miltiorrhiza flavonoid biosynthesis-related proteins and their homologs from other plant species. These genes showed tissue-specific expression patterns and differentially responded to MeJA treatment. Through comprehensive and systematic analysis, fourteen genes most likely to encode flavonoid biosynthetic enzymes were identified. The results provide valuable information for understanding the biosynthetic pathway of flavonoids in medicinal plants.
... A section of the sidewall of the enzyme's active site made up the GFGPG loop. The structure of the HsCHS deduce amino acid sequence is in agreement with the studies of Jiang et al. (2008), and Suh et al. (2000). This Gly-rich loop is known as the tag sequence of the CHS enzyme. ...
... Pro 375 of this motif is highly conserved in all the members of the CHS superfamily. This characteristic residue is unique to chalcone synthase superfamily and not for any other condensing enzymes (Suh et al. 2000). The Leucine zipper motif responsible for dimerization is another important motif in CHS proteins structure (Sderman et al. 1994). ...
Flavonoids are polyphenolic compounds commonly found in vegetables as well as fruits and contribute significantly in the human diet. The calyx of roselle (Hibiscus sabdariffa L.) is rich in polyphenolic compounds and anthocyanins. Chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) are two important genes involved in the biosynthesis of flavonoids including anthocyanins in plants. The two transcripts designated as HsCHS (KR709156) and HsF3H (KR709157) were isolated from the calyx tissue of roselle using the Rapid Amplification of cDNA Ends PCR and PCR walking approaches, which encoded the polypeptides of 389 and 368 amino acids, respectively. Several important domains were revealed in the HsCHS amino acid sequence, including CHS-like, fabH, BcsA, Chal-sti-synt-N and Chal-sti-synt-C, which indicates that the isolated gene is probably a CHS belonging to the polyketide synthase family. On the other hand, identification of 2OG-FeII_oxy, Isopenicillin N synthase-like, DIOXN and PLN02515 domains in HsF3H protein sequence supports the idea that the isolated gene is an F3H related to the large gene family of 2-oxoglutarate-dependent dioxygenases. This study predicted the putative functions of the two central genes governing the flavonoid pathway in H. sabdariffa, which leads to anthocyanin production.
... Although these differences of the MdCHSs, are at sites which are not in the highly conserved active-site amino acids (Supplemental Fig. S2). Site-directed mutagenesis revealed that this motif (GFGPG) is important for catalytic activities for CHS (Suh et al., 2000). ...
Apple (Malus x domestica Brokh.) is a widely cultivated deciduous tree species of significant economic importance. Apple leaves accumulate high levels of flavonoids and dihydrochalcones, and their formation is dependent on enzymes of the chalcone synthase family. Three CHS genes were cloned from apple leaves and expressed in Escherichia coli. The encoded recombinant enzymes were purified and functionally characterized. In-vitro activity assays indicated that MdCHS1, MdCHS2 and MdCHS3 code for proteins exhibiting polyketide synthase activity that accepted either p-dihydrocoumaroyl-CoA, p-coumaroyl-CoA, or cinnamoyl-CoA as starter CoA substrates in the presence of malonyl-CoA, leading to production of phloretin, naringenin chalcone, and pinocembrin chalcone. MdCHS3 coded a chalcone-dihydrochalcone synthase enzyme with narrower substrate specificity than the previous ones. The apparent Km values of MdCHS3 for p-dihydrocoumaryl-CoA and p-coumaryl-CoA were both 5.0 μM. Expression analyses of MdCHS genes varied according to tissue type. MdCHS1, MdCHS2 and MdCHS3 expression levels were associated with the levels of phloretin accumulate in the respective tissues.
... However, changes at T132, M137, T197, and G372FGPG were detected in the putatively encoded NtCHS6 protein (Fig. 2). T132, M137 and the G372FGPG loop are involved in cyclization reactions (Ferrer et al. 1999;Jez et al. 2000;Suh et al. 2000). The topology of the cyclization pocket controls the chalcone formation in CHS. ...
Chalcone synthases (CHS, EC 2.3.1.74) are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL (Y14507) gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress. Our results suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and might play important roles in ultraviolet protection and drought tolerance.
... In addition, a single change of His to Glu at position 166 alters the substrate preference of AhSTS from p-coumaroyl-CoA to cinnamoyl-CoA (Schröder and Schröder, 1992). But current efforts in CHS and STS conversion only partially alter the catalytic reactions through active sites or their geometry (Tropf et al., 1995;Suh et al., 2000). More evidence is needed for these CHS and STS enzymes, even with the help of the crystalline structures of STS (Shomura et al., 2005). ...
... This is consistent with the fact that changes in only a few amino acids were enough to change the qualitative enzyme functions from 2-PS or ACS to CHS (Jez et al., 2000;Abe et al., 2007). Although current experimental efforts concerning CHS and STS functions only quantitatively exchanged their substrate preferences (Schröder and Schröder, 1992;Suh et al., 2000). Attempts to exchange functions between CHS and STS enzymes requires more investigation. ...
Polyketide synthases (PKSs) utilize the products of primary metabolism to synthesize a wide array of secondary metabolites in both prokaryotic and eukaryotic organisms. PKSs can be grouped into three distinct classes, types I, II, and III, based on enzyme structure, substrate specificity, and catalytic mechanisms. The type III PKS enzymes function as homodimers, and are the only class of PKS that do not require acyl carrier protein. Plant type III PKS enzymes, also known as chalcone synthase (CHS)-like enzymes, are of particular interest due to their functional diversity. In this study, we mined type III PKS gene sequences from the genomes of six aquatic algae and 25 land plants (1 bryophyte, 1 lycophyte, 2 basal angiosperms, 16 core eudicots, and 5 monocots). PKS III sequences were found relatively conserved in all embryophytes, but not exist in algae. We also examined gene expression patterns by analyzing available transcriptome data, and identified potential cis-regulatory elements in upstream sequences. Phylogenetic trees of dicots angiosperms showed that plant type III PKS proteins fall into three clades. Clade A contains CHS/STS-type enzymes coding genes with diverse transcriptional expression patterns and enzymatic functions, while clade B is further divided into subclades b1 and b2, which consist of anther-specific CHS-like enzymes. Differentiation regions, such as amino acids 196-207 between clades A and B, and predicted positive selected sites within α-helixes in late appeared branches of clade A, account for the major diversification in substrate choice and catalytic reaction. The integrity and location of conserved cis-elements containing MYB and bHLH binding sites can affect transcription levels. Potential binding sites for transcription factors such as WRKY, SPL, or AP2/EREBP may contribute to tissue- or taxon-specific differences in gene expression. Our data shows that gene duplications and functional diversification of plant type III PKS enzymes played a critical role in the ancient conquest of the land by early plants and angiosperm diversification.
