Functional characterization of recombinant hyoscyamine 6¦Â-hydroxylase from Atropa belladonna

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• 作者：Jing Li ; Marco J. van Belkum ; John C. Vederas
• 关键词：Hyoscyamine 6¦Â-hydroxylase ; Atropa belladonna ; (&minus ; )-Hyoscyamine ; 6¦Â-Hydroxyhyoscyamine ; Scopolamine ; Tropane alkaloids
• 刊名：Bioorganic and Medicinal Chemistry
• 出版年：2012
• 出版时间：15 July, 2012
• 年：2012
• 卷：20
• 期：14
• 页码：4356-4363
• 全文大小：693 K

文摘

(?)-Hyoscyamine, the enantiomerically pure form of atropine, and its derivative scopolamine are tropane alkaloids that are extensively used in medicine. Hyoscyamine 6¦Â-hydroxylase (H6H, EC 1.14.11.11), a monomeric ¦Á-ketoglutarate dependent dioxygenase, converts (?)-hyoscyamine to its 6,7-epoxy derivative, scopolamine, in two sequential steps. In this study, H6H of Atropa belladonna (AbH6H) was cloned, heterologously expressed in Escherichia coli, purified and characterized. The catalytic efficiency of AbH6H, especially for the second oxidation, was found to be low, and this may be one of the reasons why Atropa belladonna produces less scopolamine than other species in the same family. 6,7-Dehydrohyoscyamine, a potential precursor for the last step of epoxidation, was shown not to be an obligatory intermediate in the biosynthesis of scopolamine using purified AbH6H with an in vitro ¹⁸O labeling experiment. Moreover, the nitrogen atom in the tropane ring of (?)-hyoscyamine was found to play an important role in substrate recognition.