Molecular cloning, expression and characterization of hyoscyamine 6beta-hydroxylase from hairy roots of Anisodus tanguticus.
Anisodus tanguticus, one of the indigenous Chinese ethnological medicinal plants of the Solanaceae, produces anticholinergic alkaloids such as hyoscyamine, 6 beta-hydroxyhyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase (H6H), a key enzyme in the biosynthetic pathway of scopolamine, catalyzes the hydroxylation of hyoscyamine and epoxide formation from 6 beta-hydroxyhyoscyamine to generate scopolamine. A full-length cDNA of H6H has been isolated from A. tanguticus hairy roots. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1035 bp encoding 344 amino acids with high homology to other known H6Hs. The equivalent amino acid sequence shows a typical motif of 2-oxoglutarate-dependent dioxygenase. The A. tanguticus H6H was expressed in Escherichia coli and purified for enzyme function analysis. This study characterized the recombinant AtH6H and showed it could generate scopolamine from hyoscyamine.
Liu
Tao
T
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, #1 Xian Nong Tan Street, Beijing 100050, P. R. China.
Zhu
Ping
P
Cheng
Ke-di
KD
Meng
Chao
C
He
Hui-Xia
HX
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008517:Phytotherapy; D018517:Plant Roots; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012601:Scopolamine / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D019657:Solanaceae / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012601:Scopolamine; D006899:Mixed Function Oxygenases; C052273:hyoscyamine (6S)-dioxygenase