Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna.
(-)-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.
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Jing
J
Li
Marco J
MJ
van Belkum
John C
JC
Vederas
D001532:Atropa belladonna / Q000201:enzymology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012601:Scopolamine / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D010940:Plant Proteins; D011994:Recombinant Proteins; D012601:Scopolamine; D006899:Mixed Function Oxygenases; C052273:hyoscyamine (6S)-dioxygenase