Molecular cloning and functional analysis of bergaptol-O-methyltransferase from Angelica dahurica (Bai Zhi) and using it to efficiently produce bergapten in E. coli

Abstract

Bai Zhi (Angelica dahurica), a Chinese herb, has long been used as a face cream for skin-whitening purposes. One of the known skin-whitening components, 8-hydroxybergapten is a hydroxylated product of bergapten that is converted from bergaptol by bergaptol 5-O-methyltransterase (BMT) in Bai Zhi. The complementary DNA of BMT was cloned from Bai Zhi root using a pair of degenerate primers designed from the highly conserved regions of other plant O-methyltransferases (OMTs). RT-PCR analysis indicated that a single band of DNA fragment corresponding to AdBMT sequence was obtained. The tandem 5'- and 3'-rapid amplification of cDNA ends via polymerase chain reaction was used to obtain the full-length cDNA sequences. The AdBMT cDNA contains an open reading frame of 1,080 bp encoding a BMT polypeptide of 359 amino acids with a calculated molecular mass of 39 kDa and a calculated pI of 5.9. Sequence alignment revealed the considerable sequence similarity of AdBMT to those of other plant OMTs. The AdBMT sequence contains conserved region I-V, similar to other plant OMTs. His-tagged AdBMT was expressed in E. coli and partially purified by ammonium sulfate precipitation. The recombinant AdBMT is most active in potassium phosphate buffer at pH 7.5 and 35 degrees C. The enzyme does not require a divalent cation for activity and the addition of Cu2+, Ni2+, and Co2+ at concentrations even as low as 0.1 mM severely inhibits enzyme activity. A simple and efficient production of bergapten in the E. coli culture overexpressing AdBMT was performed. The bergapten yield is approximately 13-fold higher than that produced by enzymes in the ammonium sulfate-purified fraction. With the supply of bergaptol in the medium, E. con cells can be used as a potential bioreactor to produce bergapten