Article

Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna.

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Bioorganic & medicinal chemistry (impact factor: 2.82). 05/2012; 20(14):4356-63. DOI:10.1016/j.bmc.2012.05.042 pp.4356-63
Source: PubMed

ABSTRACT (-)-Hyoscyamine, the enantiomerically pure form of atropine, and its derivative scopolamine are tropane alkaloids that are extensively used in medicine. Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), a monomeric α-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 (18)O labeling experiment. Moreover, the nitrogen atom in the tropane ring of (-)-hyoscyamine was found to play an important role in substrate recognition.

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Keywords

18)O labeling experiment
 
6,7-Dehydrohyoscyamine
 
6,7-epoxy derivative
 
Atropa belladonna
 
catalytic efficiency
 
converts
 
derivative scopolamine
 
enantiomerically pure form
 
Escherichia coli
 
Hyoscyamine 6β-hydroxylase
 
last step
 
monomeric α-ketoglutarate dependent dioxygenase
 
nitrogen atom
 
obligatory intermediate
 
scopolamine
 
sequential steps
 
substrate recognition
 
tropane ring
 
vitro
 

Jing Li