Hyoscyamine 6p-hydroxylase

Matsuda, J. et al.. Molecular cloning of hyoscyamine 6p-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger. J. Biol Chem., 266, 9460, 1991. 

HASHIMOTO, T., HAYASHI, A., AMANO, Y, KOHNO, J., IWANARI, H., USUDA, S., YAMADA, Y., Hyoscyamine 6P-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root. J. Biol. Chem., 1991,266, 4648-4653. 

HASHIMOTO, T., YAMADA, Y., Purification and characterization of hyoscyamine 6P-hydroxylase from root cultures of Hyoscyamus niger L. Hydroxylase and epoxidase activities in the enzyme preparation. Eur. J. Biochem., 1987, 164, 277-286. 

Important for the production of scopolamine, hyoscyamine 6p-hydroxylase was the first enzyme of tropane alkaloid metabolism to be purified and remains the most rigorously studied. It was obtained in pure form from H. niger root cultures and the preparation showed that it is a bifunctional enzyme with activity both as the 7(3-hydroxylase and as the 6,7 3-epoxidase. A clone for H6H was obtained following the purification of enzyme activity. The gene shows some similarity to other hydroxylases, including those involved in oxidative reactions in the formation of ethylene and anthocyanins (Hashimoto and Yamada, 1994). 

Kanegae, T., Kajiya, H., Amano, Y., Hashimoto, T. and Yamada, Y. (1994) Species-dependent expression of the hyoscyamine 6p-hydroxylase gene in the pericycle. Plant Physiol, 105, 483-90. 

Both alkaloids have (+) and (-) forms but only the (-) hyoscyamine and (-) scopolamine are active. The biosynthetic pathway of tropane alkaloids, Fig. (1) is not totally understood, especially at the enzymatic level. Edward Leete has pioneered the biosynthetic studies of tropane alkaloid since 1950"s using whole plants and isotope labels [85-86]. The tropane alkaloid hyoscyamine is bioconverted by the enzyme H6H (hyoscyamine 6p-hydroxylase, EC 1.14.11.11) to scopolamine via 6p-hydroxyhyoscyamine. Hyoscyamine is the ester of tropine and (S)-tropic acid. The (S)-tropic acid moiety derives from the amino acid L-phenylalanine, while the bicyclic tropane ring derives from L-omithine primarily or L-arginine via tropinone. Tropinone is stereospecifically reduced to form either, tropine which is incorporated into hyoscyamine, or on the other hand into pseudotropine which proceeds to calystegines, a group of nortropane derivates that were first found in the Convolvulaceae family [87]. 

F. (1). Tropane alkaloids biosynthetic pathway. The known enzymes are indicated. ODC (ornithine decarboxylase), ADC (arginine decarboxylase), PMT (putrescine methyl transferase), MPO (methyl putrescine oxidase), TRI, TRII (tropinone reductase I, II), H6H (hyoscyamine 6p hydroxylase). 

Hyoscyamine 6P-hydroxylase (H6H, EC 1.14.11.11) is a 2-oxoglutarate-dependent dioxygenase and is the final enzyme of the tropane alkaloid biosynthetic pathway. The H6H catalyzes the conversion of hyoscyamine into 6P-hydoxyhyoscyamine and scopolamine by two sequential reactions [24, 25]. Firstly, it catalyses the 6P-hydroxylation of hyoscyamine and secondly, the formation of the epoxy group leading to scopolamine [13]. Several works have revealed that the epoxidation activity is much lower than the hydroxylation one [11, 26]. These findings are in agreement to those reported in our laboratory about the H6H enzyme isolated from Brugmansia Candida (syn. Datura Candida) [27-29]. This South America n native plant is a natural hybrid between B. aurea and B. versicolor and a tropane alkaloids producer [30,31]. 

Zdrate R, El Jaber-Vazdekis N, Medina B, Ravelo AG (2006) Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropa baetica by over-expressing the gene encoding hyoscyamine 6P-hydroxylase. Biotechnol Lett 28(16) 1271-1277... 

Hyoscyamine 6P - hydroxylase (HRH) Atropa belladonna, Hyoscyamus niger, Hyoscyamus muticus... 

Pramod KK, Singh S, Jayabaskaran C (2009) Expression of hyoscyamine 6P-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L. Plant Sci (Shannon, Irel) 178 (Copyright (C) 2012 American Chemical Society (ACS). All Rights Reserved.) 202-206. doi 10.1016/j.plantsci.2009.11.004... 

Yamada Y, Hashimoto T (1989) Substrate specifity of the hyoscyamine 6P-hydroxylase from cultured roots of Hyoscyamus niger. Proc Jpn Acad, Ser B Phys Biol Sci 65 156-159 Yamaguchi H, Nishimoto K (1965) Studies on the alkaloids of the root of Physalis alkekengi (I). 

Fig. 16.1 Biosynthetic pathways for nicotine euid tropane eilkaloids. Enzymes involved in the pathways are PMT putrescine Af-methyltransferase, MPO Al-methylputrescine oxidase, TR-I tropine-forming tropinone reductase, TR-II pseudotropine-forming tropinone reductase, MecgoR methylecgonone reductase, CYP80F1 cytochrome P450 monooxygenase with Uttorine mutase activity, H6H hyoscyamine 6p-hydroxylase...

Suzuki K, Yamada Y, Hashimoto T (1999a) Expression of Atropa belladonna putrescine A-methyltransferase gene in root pericycle. Plant CeU Physiol 40 289-297 Suzuki K, Yun DY, Chen XY et al (1999b) An Atropa belladonna hyoscyamine 6p-hydroxylase gene is differentially expressed in the root pericycle and anthers. Plrmt Mol Biol 40 141-152 Thompson JE, Hopkins MT, Taylor C et al (2004) Regulation of senescence by eukaryotic trrmsla-tion initiation factor 5A implications for plant growth and development. Trends Plant Sd 9 174-179... 

---