Molecular and Biochemical Characterization of an Enzyme Responsible for the Formation of Hypericin in St. John's Wort (Hypericum perforatum L.)

A major gene termed Hyp-1 encoding for hypericin (HyH) biosynthesis was cloned and characterized from Hypericum perforatum (St. John's wort) cell cultures. H. perforatum leaves are widely used as an herbal remedy in the treatment of mild to moderate depression. Hypericin, a photosensitive and red-colored naphthodianthrone, has been reported as the bioactive compound responsible for reversing the depression symptoms. In this study a novel red-color-based colony screening method for examining a cDNA library (λ-TriplEX2) derived from H. perforatum cell cultures revealed the gene responsible for hypericin biosynthesis after the administration of emodin, a precursor of hypericin. The selected clones were expressed in Escherichia coli (BM 25.8 line) and were further screened for biosynthesis of emodin to hypericin, which resulted in an 84.6% conversion. The full-length cDNA sequence of Hyp-1 is 782 nucleotides in length with an open reading frame of 477 nucleotides coding for a protein of 159 amino acids, with a 45.1% homology to Bet.v.1 class allergens. Reverse transcriptase-PCR analysis showed high levels of Hyp-1 transcripts in dark-grown cell cultures compared with the levels in light-grown cell cultures and leaves. Southern blot analysis showed the presence of a single Hyp-1 gene in H. perforatum. Furthermore, Hyp-1 was expressed with a His6 affinity tag linked to its N terminal region using the expression vector pET-28a, and the recombinant Hyp-1 protein was able to convert HyH from emodin under in vitro conditions. HyH product inhibition was observed with emodin analogues, rhein, rhein methyl ester, and DNA3-55-1. Our results demonstrate a direct and complex conversion of emodin to HyH that is solely catalyzed by Hyp-1, a Bet.v.1 class allergen from H. perforatum. A major gene termed Hyp-1 encoding for hypericin (HyH) biosynthesis was cloned and characterized from Hypericum perforatum (St. John's wort) cell cultures. H. perforatum leaves are widely used as an herbal remedy in the treatment of mild to moderate depression. Hypericin, a photosensitive and red-colored naphthodianthrone, has been reported as the bioactive compound responsible for reversing the depression symptoms. In this study a novel red-color-based colony screening method for examining a cDNA library (λ-TriplEX2) derived from H. perforatum cell cultures revealed the gene responsible for hypericin biosynthesis after the administration of emodin, a precursor of hypericin. The selected clones were expressed in Escherichia coli (BM 25.8 line) and were further screened for biosynthesis of emodin to hypericin, which resulted in an 84.6% conversion. The full-length cDNA sequence of Hyp-1 is 782 nucleotides in length with an open reading frame of 477 nucleotides coding for a protein of 159 amino acids, with a 45.1% homology to Bet.v.1 class allergens. Reverse transcriptase-PCR analysis showed high levels of Hyp-1 transcripts in dark-grown cell cultures compared with the levels in light-grown cell cultures and leaves. Southern blot analysis showed the presence of a single Hyp-1 gene in H. perforatum. Furthermore, Hyp-1 was expressed with a His6 affinity tag linked to its N terminal region using the expression vector pET-28a, and the recombinant Hyp-1 protein was able to convert HyH from emodin under in vitro conditions. HyH product inhibition was observed with emodin analogues, rhein, rhein methyl ester, and DNA3-55-1. Our results demonstrate a direct and complex conversion of emodin to HyH that is solely catalyzed by Hyp-1, a Bet.v.1 class allergen from H. perforatum. Plants have been estimated to collectively synthesize more than 100,000 different secondary metabolites, many of which have applications as medicinal compounds (1Dixon R.A. Nature. 2001; 411: 843-847Crossref PubMed Scopus (1259) Google Scholar). One such compound is hypericin (HyH), 1The abbreviations used are: HyH, hypericin; HPLC, high-performance liquid chromatography; RT, reverse transcriptase; LC-MS, liquid chromatography-mass spectrometry; PR, pathogenesis-related.1The abbreviations used are: HyH, hypericin; HPLC, high-performance liquid chromatography; RT, reverse transcriptase; LC-MS, liquid chromatography-mass spectrometry; PR, pathogenesis-related. a naphthodianthrone produced in the dorsal leaf glands of Hypericum perforatum L., commonly known as St. John's wort (2Deltito J. Beyer D. J. Affect. Disord. 1998; 51: 245-251Crossref Scopus (58) Google Scholar). Standardized extracts of St. John's wort containing 3% HyH have been successfully tested in numerous studies for the treatment of mild to moderate depression (2Deltito J. Beyer D. J. Affect. Disord. 1998; 51: 245-251Crossref Scopus (58) Google Scholar, 3American Botanical CouncilTherapeutic Monographs on Medicinal Plants for Human Use. Austin, TX1998: 256-298Google Scholar). HyH, a red naphthodianthrone, has been reported as the active compound found in St. John's wort and has been shown to act as a monoamine oxidase inhibitor (4Linde K. Ramirez G. Mulrow C.D. Br. Med. J. 1996; 313: 253-258Crossref PubMed Scopus (937) Google Scholar). As is true for most anthraquinones, there are no published descriptions of the biosynthetic pathway that leads to HyH production. The majority of commercially available St. John's wort formulations are crude extracts containing minor proportions of HyH combined with flavonoids, phenols, tannins, and volatile oils, which may account for some of the reported side effects of the extracts (5Vogel G. Science. 2001; 291: 35-37Crossref PubMed Scopus (17) Google Scholar, 6Butterweck V. Bockers T. Korte B. Wittkowski W. Winterhoff H. Brain Res. 2002; 930: 21-29Crossref PubMed Scopus (98) Google Scholar), such as nausea and vomiting. These side effects are more severe than those encountered with synthetic antidepressants (4Linde K. Ramirez G. Mulrow C.D. Br. Med. J. 1996; 313: 253-258Crossref PubMed Scopus (937) Google Scholar). Studies have shown that St. John's wort formulations may activate the cytochrome P450 (CYP3A) pathway, accelerating the breakdown of other drugs (5Vogel G. Science. 2001; 291: 35-37Crossref PubMed Scopus (17) Google Scholar, 7Bray B.J. Brennan N.J. Perry N. Menkes D.B. Rosengren R.J. Life Sci. 2002; 70: 1325-1335Crossref PubMed Scopus (22) Google Scholar). For instance, the blood concentrations of the anti-clotting drug warfarin were dramatically reduced when taken together with St. John's wort extracts (5Vogel G. Science. 2001; 291: 35-37Crossref PubMed Scopus (17) Google Scholar, 8Schulz V. Phytomedicine. 2001; 8: 152-160PubMed Google Scholar). Similar drug dilution cases were observed with birth control pills and coronary transplant anti-rejection medications (5Vogel G. Science. 2001; 291: 35-37Crossref PubMed Scopus (17) Google Scholar). Recent reports indicate some metabolites other than HyH as the cause of these unwanted drug interactions in St. John's wort extracts (5Vogel G. Science. 2001; 291: 35-37Crossref PubMed Scopus (17) Google Scholar, 9Kientsch U. Burgi S. Ruedeberg C. Pharmacopsychiatry. 2001; 34: 56-60Crossref Scopus (35) Google Scholar). Thus, the purification and bioprocessing of HyH, the antidepressant compound in St. John's wort, may be of medical importance in the treatment of depression, and in avoiding the side effects associated with this herbal remedy. Little is known about the biosynthesis of hypericin other than that it lies on the polyketide pathway, presumably through emodin (Fig. 1) (10Mazur, Y., Bock, H., and Lavie, D. (1992) United States Patent 0432496.Google Scholar), and with protohypericin as the penultimate precursor (11Leistner A.G. Photochem. Photobiol. 1971; 27: 347-349Google Scholar). Synthetically, it is readily prepared by treating emodin dianthrone with ferrous sulfate and pyridine-N-oxide in pyridine, followed by light irradiation of the formed protohypericin (10Mazur, Y., Bock, H., and Lavie, D. (1992) United States Patent 0432496.Google Scholar, 11Leistner A.G. Photochem. Photobiol. 1971; 27: 347-349Google Scholar). This synthesis could be in part a biomimetic one. However, nothing is known about the sequence of steps in H. perforatum or other plants that lead to protohypericin and finally to HyH, and no enzymes or genes in this pathway have been purified or cloned. We developed an approach to identify the gene(s) responsible for the biosynthesis of HyH using a cDNA library constructed from H. perforatum cell cultures, supplemented with emodin (a possible precursor for HyH) and using a novel color-based screening methodology. Biochemical characterization of the recombinant protein revealed saturation kinetics for different concentrations of emodin. Other physiological factors such as the effect of divalent ions, ATP, temperature, and pH were also analyzed for their influence on enzyme activity. Our results also show variable product inhibition patterns by different structure analogues of emodin. Our studies show that the gene involved in HyH biosynthesis from emodin has a close homology to Bet.v.1 class allergens and that its enzymatic activity may involve a condensation followed by dehydration and two oxidative coupling reactions. Seeds of H. perforatum L. (S5195) were obtained from Richters, Ontario, Canada. Plant growth regulators were obtained from Sigma Co. (St. Louis, MO). Emodin structure analogues, rhein and rhein methyl ester were from the Stermitz laboratory, Department of Chemistry, Colorado State University, Fort Collins, CO, and DNA3-55-1 was obtained from Dr. Juanne Romagni, University of St. Thomas, Texas. All other chemicals were of analytical and HPLC grade and were obtained from Sigma Co. unless specified. Callus cultures were initiated using leaves from a 10-day-old H. perforatum plant grown in vitro as previously described by Bais et al. (12Bais H.P. Walker T.S. McGrew J.J. Vivanco J.M. In Vitro Cell. Dev. Biol.-Plant. 2002; 38: 58-65Crossref Scopus (42) Google Scholar). Cell suspension cultures were established from the callus cultures and were maintained in 125-ml Erlenmeyer flasks with 50 ml of nutrient MS (13Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (52758) Google Scholar) medium containing 2,4-dichlorophenoxyacetic acid (0.90 μm), kinetin (0.11 μm), and sucrose (30 g liter–1) by biweekly sub-culturing on a rotary shaker at 90 rpm maintained at 25 ± 2 °C. Cell cultures of 1-month-old H. perforatum plants were collected and the total soluble proteins from the roots were extracted as described before by Vivanco et al. (14Vivanco J.M. Savary B.J. Flores H.E. Plant Physiol. 1999; 119: 1447-1456Crossref PubMed Scopus (88) Google Scholar). Different concentrations of total proteins (0.5–30 and emodin (0.5–30 were with a of and pH for at under conditions. The was extracted with and the were to under The obtained after of emodin with the total cell proteins was further extracted using ml of and was for The was at rpm for The was further by and the was in ml of The were prepared for analysis after through a were by on a The of to an The at was by a variable A of a of to pH with and was and A was for HyH as described by Bais et al. (12Bais H.P. Walker T.S. McGrew J.J. Vivanco J.M. In Vitro Cell. Dev. Biol.-Plant. 2002; 38: 58-65Crossref Scopus (42) Google Scholar), with an of and a of ml HyH was at HyH was from Sigma and were compared with the HyH on the of and was used for The was an at a of The MS were using HyH as the high and were to the HyH after was used for to a of were used for A of and a of was used the Our studies indicate that of H. perforatum grown in in of HyH as compared with grown under light (12Bais H.P. Walker T.S. McGrew J.J. Vivanco J.M. In Vitro Cell. Dev. Biol.-Plant. 2002; 38: 58-65Crossref Scopus (42) Google Scholar, T.S. Bais H.P. Vivanco J.M. 2002; PubMed Scopus Google for the study dark-grown of H. perforatum were used for cDNA library was extracted from cell suspension cultures of H. perforatum. H. perforatum were in liquid and the total was extracted using an of total was used for cDNA synthesis using reverse The cDNA was by of The was with and a vector using the cDNA The vector was an using the A vector containing the H. perforatum cDNA library was with Escherichia coli cell line) for The of recombinant clones was by screening in the presence of and screening of the library for genes involved in the biosynthesis of HyH was after at under by emodin on the and the that emodin red-colored were selected and after screening of was to the screening by of emodin to the grown which red were with a and to in Escherichia coli (BM by in and a from the recombinant For the conversion of emodin to HyH, selected coli clones were under in J. PubMed Google Scholar) ml in a Erlenmeyer emodin was to and the HyH using as described (BM and selected from secondary and screening were using a The with the cDNA were used to for the of The and obtained from secondary and were extracted as the described and analyzed by for HyH was prepared using a and used for University of The sequence was followed by amino acid to proteins using The for the was obtained by with from a full-length sequence from the 25.8 The product was on and extracted from the using a The was with using from the This sequence was used as a for was extracted from in vitro grown plants using a of total was using different enzymes and The were on to and with a sequence obtained by an Plant extracted the total from and grown in and from and dark-grown cell suspension cultures. of was used for cDNA synthesis using a cDNA synthesis Reverse was to the for the cDNA using a of to the control Hyp-1 and a of to the gene for in the different the of was to through an in were using an Hyp-1 protein was expressed with His6 affinity tag linked to its N region using the expression vector the full-length Hyp-1 was constructed by using the a and a The product was cloned and the was using a from a single The Hyp-1 sequence was from the with and and cloned with the The was for protein expression grown was for gene expression with for and the proteins were extracted by The recombinant Hyp-1 protein was purified using using an The was to the in and and with the to The was with and to proteins that were the recombinant Hyp-1 protein was from the with and The of the recombinant Hyp-1 protein was analyzed by of the on The recombinant Hyp-1 protein was with emodin using the described and HyH biosynthesis was analyzed by for HyH for HyH was in of of pH containing different concentrations of precursor at 25 °C. The enzymatic activity was after the of of recombinant Hyp-1 protein an of The conversion to HyH was by a at The activity was expressed in of HyH produced activity were in The and for the HyH were under the described All and were by analysis and in and effect of divalent for HyH at concentrations of precursor emodin was by with μm), and to the For studies was in the and at 25 for of recombinant Hyp-1 enzyme was obtained by the enzymatic activity in of of pH containing different concentrations of precursor with were as described The pH of the recombinant enzyme was obtained by the enzymatic activity in of of containing different concentrations of emodin with pH was as described analogues for emodin, rhein, and rhein methyl ester were prepared at concentrations of in pH The effect of the analogues was at concentrations of emodin by concentrations of the All with rhein, and rhein methyl ester were with of recombinant Hyp-1 with an of The product was as described for HyH in Cell of H. steps in the biosynthesis of HyH are derived from and (10Mazur, Y., Bock, H., and Lavie, D. (1992) United States Patent 0432496.Google Scholar). These steps are followed by steps to the of an which may further to emodin and emodin (Fig. involved many steps and HyH could be prepared with a from emodin which is available from commercially available emodin (10Mazur, Y., Bock, H., and Lavie, D. (1992) United States Patent 0432496.Google Scholar). We that in H. perforatum conversion of emodin to HyH may be to an enzymatic In of this found of emodin in H. perforatum cell cultures of Emodin with from Cell of H. in vitro with emodin and total proteins from dark-grown cell cultures of H. perforatum. Emodin with of total protein extracts resulted in its to HyH (Fig. A and We the of and total protein with the of the results of emodin (0.5–30 to the total protein extracts (0.5–30 from cell cultures. Emodin with of total protein extracts resulted in the to HyH (Fig. The presence of HyH was further by MS analysis (Fig. These results the presence of enzymatic activity in the protein which catalyzed HyH biosynthesis from emodin under conditions. cDNA and of cDNA library was constructed by from dark-grown cell suspension cultures of H. perforatum using the screening of a cDNA library was from the library using coli cell a at were selected by screening to and selected clones red The were and used for in coli (BM cell of and to 25.8 cell formed red-colored emodin administration under (Fig. The red were and for using the of the clones showed a of analysis and clones in 25.8 were tested for using was observed that clones showed an conversion from emodin to HyH compared with the control 25.8 cell the (Fig. A and showed the HyH conversion compared with the other clones (Fig. it was observed that the resulted in the conversion of emodin to HyH at levels under (Fig. coli (BM the and medium convert to HyH administration (Fig. The containing cDNA was prepared using a and was used for and to Bet.v.1 The cDNA of Hyp-1 sequence has with an open reading frame of nucleotides coding for a protein of 159 amino acid (Fig. A and All clones analyzed a region of nucleotides the and a the cloned gene is responsible for biosynthesis of HyH, it was termed A using the amino acid sequence of Hyp-1 produced with the of proteins found in a of (Fig. sequence revealed homology Hyp-1 and a major allergen with a 45.1% followed by a major allergen D. U. B. K. D. S. 2002; 38: PubMed Scopus Google Scholar) with in amino acid sequence to Hyp-1 (Fig. Hyp-1 showed homology with other of a major allergen S. K. D. S. 34: PubMed Scopus Google Scholar), Bet.v.1 from K. C. J. F. H. PubMed Scopus Google Scholar), and a allergen C. D. Plant Physiol. 1999; PubMed Scopus Google Scholar) (Fig. The amino acid sequence analysis that Hyp-1 has a of with a of The for the sequence of Hyp-1 with the Bet.v.1 The which is a of is at amino acid in of Hyp-1 in H. analysis of the total from different of H. and cell cultures, that this gene is the with levels in dark-grown cell cultures of H. perforatum (Fig. The recombinant coli also showed the of transcripts for Hyp-1 (Fig. As observed Hyp-1 gene expression was in under compared with light-grown and in vitro grown leaves (Fig. (12Bais H.P. Walker T.S. McGrew J.J. Vivanco J.M. In Vitro Cell. Dev. Biol.-Plant. 2002; 38: 58-65Crossref Scopus (42) Google Scholar, T.S. Bais H.P. Vivanco J.M. 2002; PubMed Scopus Google Scholar). Southern study the of genes to Hyp-1 in H. was with and on and of the enzymes used for the Hyp-1 cDNA The blot was with Hyp-1 A single in these All the show with the could be to with the Hyp-1 The of H. perforatum with showed a single in that Hyp-1 gene in H. perforatum (Fig. of Hyp-1 in recombinant Hyp-1 protein expressed in coli was and for on a which showed a single protein of (Fig. This protein is in with the amino acid sequence analysis that that Hyp-1 has a of The possible in Hyp-1 could be to the of the His6 tag amino at the region of Hyp-1 protein from the was observed that the recombinant Hyp-1 protein showed an conversion from emodin to HyH under in vitro as and analyzed by analysis (Fig. and Biochemical of enzymatic activity of the purified recombinant enzyme was by the concentrations of precursor emodin. A was for analysis of HyH The of HyH a at (Fig. The of enzymatic activity as by product (HyH) was to the concentrations of the precursor (Fig. The and were obtained by of HyH with emodin as a The and were and of HyH (Fig. The product using emodin at different showed an activity at (Fig. The enzyme of its activity at (Fig. The enzymatic activity of recombinant Hyp-1 was at pH and at pH (Fig. a in enzyme with However, the recombinant enzyme was also to pH in the (Fig. protein Hyp-1 showed high to HyH at 25 and (Fig. and the activity at (Fig. The recombinant protein Hyp-1 was by the of divalent and in the (Fig. Hyp-1 showed some on for its activity to HyH (Fig. The activity was obtained at in a (Fig. of physiological factors on recombinant Hyp-1 activity using different concentrations of emodin in for at 25 °C. The conversion to HyH was by a at The activity is expressed in effect of different concentrations supplemented with known concentrations of emodin and recombinant effect of different levels supplemented with emodin and recombinant effect of different levels supplemented with known concentrations of emodin and recombinant effect of different concentrations supplemented with known concentrations of emodin and recombinant effect of different pH supplemented with known concentrations of emodin and recombinant effect of different supplemented with known concentrations of emodin and recombinant The obtained are of of of the purified recombinant protein Hyp-1 was with structure analogues of emodin such as rhein, rhein methyl ester, and DNA3-55-1 (Fig. The product inhibition of structure analogues of emodin was observed with the DNA3-55-1 (Fig. with inhibition of HyH observed with DNA3-55-1 (Fig. inhibition of HyH when DNA3-55-1 was to the (Fig. The product inhibition was reduced with anthraquinones, rhein, and rhein methyl ester than with DNA3-55-1 (Fig. rhein, and rhein methyl ester showed product inhibition at and (Fig. concentrations of rhein and rhein methyl ester resulted in and in product (Fig. The of HyH from H. perforatum is on a of by other metabolites and the of it from the (10Mazur, Y., Bock, H., and Lavie, D. (1992) United States Patent 0432496.Google Scholar, 11Leistner A.G. Photochem. Photobiol. 1971; 27: 347-349Google Scholar). show the direct conversion of emodin to HyH by Hyp-1, a gene from dark-grown cell cultures of H. perforatum. The amino acid sequence of Hyp-1 has a close homology with the Bet.v.1 allergen Bet.v.1 is a major allergen from the to the Bet.v.1 are the cause of H. H. J. Scopus Google Scholar, S. S. B. N. Sci. 2002; PubMed Scopus Google Scholar), and this protein homology to proteins K. PubMed Scopus Google Scholar, H. C. J. PubMed Scopus Google Scholar, C. D. H. Res. PubMed Scopus Google Scholar), which are expressed in plant and treatment H. Plant Cell Scopus Google Scholar, J. Plant PubMed Scopus Google Scholar). The amino acid sequence for Hyp-1 of the of Bet.v.1 class allergens K. C. J. F. H. PubMed Scopus Google Scholar). A in Hyp-1 is the from the Bet.v.1 as as in the proteins have been to have and Scopus Google Scholar, G. J.J. PubMed Scopus Google Scholar). In a study it was shown that Bet.v.1 has an affinity to a of which the plant and J. 2002; PubMed Scopus Google Scholar). was that the class allergen to as showed close homology to as as to F. H. D. S. J. 2001; PubMed Scopus Google Scholar, H. K. G. W. J. 1999; PubMed Scopus Google Scholar). Recent studies on recombinant and a protein from have shown that these proteins convert to by activity F. H. D. S. J. 2001; PubMed Scopus Google Scholar). and a single the from and to and in the and biosynthetic F. H. D. S. J. 2001; PubMed Scopus Google Scholar, H. K. G. W. J. 1999; PubMed Scopus Google Scholar). as as Bet.v.1 class allergens have a in cell no enzymatic activity has been to this Plant PubMed Scopus Google Scholar). The expression of the Bet.v.1 class allergens is and the of these proteins is HyH was at a of in H. perforatum cell cultures (12Bais H.P. Walker T.S. McGrew J.J. Vivanco J.M. In Vitro Cell. Dev. Biol.-Plant. 2002; 38: 58-65Crossref Scopus (42) Google Scholar, T.S. Bais H.P. Vivanco J.M. 2002; PubMed Scopus Google Scholar). However, the HyH was shown to be a in H. perforatum (12Bais H.P. Walker T.S. McGrew J.J. Vivanco J.M. In Vitro Cell. Dev. Biol.-Plant. 2002; 38: 58-65Crossref Scopus (42) Google Scholar) that the of emodin, and its to Hyp-1, levels of Hyp-1 activity pH and or as reported for other enzymes F. H. D. S. J. 2001; PubMed Scopus Google Scholar, H. K. G. W. J. 1999; PubMed Scopus Google Scholar), and a 25 and that the enzyme is resulted in of the enzymatic activity compared with other tested divalent The enzyme and of compounds in the emodin conversion was tested using structure analogues of emodin. The product inhibition activity of DNA3-55-1 that the side of this emodin and its of it compared with other This could be for The that the Hyp-1 sequence showed a close homology to Bet.v.1 class allergens a novel enzymatic for this of Hyp-1 a direct and of emodin to HyH in the This may involve a condensation followed by dehydration and a sequence of oxidative to We that Hyp-1 an condensation emodin and emodin followed by dehydration to emodin Emodin dianthrone may to which in This is were under it that Hyp-1 may be involved in this oxidative on results that Hyp-1 may at a condensation followed by dehydration and two oxidative coupling reactions. We Dr. for of rhein methyl

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