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

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• 作者：K.K. Pramod ; Satpal Singh ; C. Jayabaskaran
• 关键词：Datura metel ; Enzyme kinetics ; Hyoscyamine ; Hyoscyamine 6β ; -hydroxylase ; Scopolamine ; Tropane alkaloids
• 刊名：Plant Physiology and Biochemistry
• 出版年：2010
• 出版时间：December 2010
• 年：2010
• 卷：48
• 期：12
• 页码：966-970
• 全文大小：351 K

文摘

Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-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. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-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 ;c;M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-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.