Molecular cloning, expression and characterization of hyoscyamine 6β-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.