Publications

• Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger.

  Matsuda J., Okabe S., Hashimoto T., Yamada Y.

  Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on t ... >> More

  Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger. << Less

  J. Biol. Chem. 266:9460-9464(1991) [PubMed] [EuropePMC]

• Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures.

  Hashimoto T., Yamada Y.

  Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6beta-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6beta-hydroxylase ... >> More

  Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6beta-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6beta-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe(2+) ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-(14)C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO(2) from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6beta-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures. << Less

  Plant Physiol 81:619-625(1986) [PubMed] [EuropePMC]