Biochemical and structural characterization of recombinant hyoscyamine 6 beta-hydroxylase from Datura metel L.

Pramod, KK and Singh, Satpal and Jayabaskaran, C (2010) Biochemical and structural characterization of recombinant hyoscyamine 6 beta-hydroxylase from Datura metel L. In: Plant Physiology and Biochemistry, 48 (12). pp. 966-970.

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Abstract

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6 beta-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. mete! (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. mete! (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6 beta-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K-m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 mu M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of alpha-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M-1 and 0.56 M-1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Item Type:	Journal Article
Publication:	Plant Physiology and Biochemistry
Publisher:	Elsevier Science
Additional Information:	Copyright of this article belongs to Elsevier Science.
Keywords:	Datura metel; Enzyme kinetics; Hyoscyamine; Hyoscyamine 6 beta-hydroxylase; Scopolamine; Tropane alkaloids
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	21 Dec 2010 08:42
Last Modified:	21 Dec 2010 08:42
URI:	http://eprints.iisc.ac.in/id/eprint/34551

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