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 (lambda-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.
Bais
Harsh Pal
HP
Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, 80523-1173, USA.
Vepachedu
Ramarao
R
Lawrence
Christopher B
CB
Stermitz
Frank R
FR
Vivanco
Jorge M
JM
D000595:Amino Acid Sequence; D000873:Anthracenes; D001483:Base Sequence; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D018076:DNA, Complementary; D020902:Hypericum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010569:Perylene / Q000031:analogs & derivatives* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D000873:Anthracenes; D018076:DNA, Complementary; D010940:Plant Proteins; D010569:Perylene; C004965:hypericin