Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina

Plant cell suspension cultures of Rauwolfia produce within 1 week similar t o 250 nkat/l of raucaffricine-O-beta-D-glucosidase. A five step procedure u sing anion exchange chromatography, chromatography on hydroxylapatite, gel filtration and FPLC-chromatography on Mono Q and Mono P delivered in a yiel d of 0.9% similar to 1200-fold enriched glucosidase. A short protocol emplo ying DEAE sepharose, TSK 55 S gel chromatography and purification on Mono Q gave a 5% recovery of glucosidase which was 340-fold enriched. SDS-PAGE sh owed a M-r for the enzyme of 61 kDa. The enzyme is not glycosylated. Struct ural investigation of the enzyme product, vomilenine, demonstrated that the alkaloid exists in aqueous solutions in an equilibrium of 21(R)- and 21(S) -vomilenine in a ratio of 3.4:1. Proteolysis of the pure enzyme with endopr oteinase Lys C revealed six peptide fragments with 6-24 amino acids which w ere sequenced. The two largest fragments showed sequences, of which the mot if Val-Thr-Glu-Asn-Gly is typical for beta-glucosidases. Sequence alignment of these fragments demonstrated high homologies to linamarase from Manihot esculenta (81% identity) or to beta-glucosidase from Prunus avium (79% ide ntity). Raucaffricine- O-beta-D-glucosidase seems to be a new member of the family 1 of glycosyl hydrolases. (C) 1999 Elsevier Science Ltd. All rights reserved.