Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis.
Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.
Warzecha
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Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Johannes Gutenberg-Universität Mainz, Germany.
Gerasimenko
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I
Kutchan
T M
TM
Stöckigt
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J
D000470:Alkaloids / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005959:Glucosidases / Q000235:genetics* / Q000378:metabolism; D026121:Indole Alkaloids; D019684:Magnoliopsida / Q000201:enzymology*; D008969:Molecular Sequence Data; D046948:Secologanin Tryptamine Alkaloids / Q000378:metabolism; D013379:Substrate Specificity
D000470:Alkaloids; D018076:DNA, Complementary; D026121:Indole Alkaloids; D046948:Secologanin Tryptamine Alkaloids; C119909:vomilenine; D005959:Glucosidases; C119910:raucaffricine beta-glucosidase