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 w hich produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whe reas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkal oid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase, known as raucaffricine-O-beta-D-glucosidase (RG), which catal yzes the in vitro formation of vomilenine, a direct intermediate in ajmalin e biosynthesis. Here, we describe the molecular cloning and functional expr ession of this enzyme in Escherichia coli. RG shows up to 60% amino acid id entity with other glucosidases of plant origin and it shares several sequen ce motifs with family 1 glucosidases which have been characterized. The bes t substrate specificity for recombinant RG was raucaffricine (K-M 1.3 mM, V -max 0.5 nkat/mu g protein) and only a few closely related structural deriv atives were also hydrolyzed. Moreover, an early intermediate of ajmaline bi osynthesis, strictosidine; is a substrate for recombinant RG (K-M 1.8 mM, V -max 2.6 pkat/mu g protein) which was not observed for the low amounts of e nzyme isolated from Rauvolfia cells. (C) 2000 Elsevier Science Ltd. All rig hts reserved.