Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia.

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.

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 approximately 250 nkat/l of raucaffricine-O-beta-D-glucosidase. A five step procedure using anion exchange chromatography, chromatography on hydroxylapatite, gel filtration and FPLC-chromatography on Mono Q and Mono P delivered in a yield of 0.9% approximately 1200-fold enriched glucosidase. A short protocol employing 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 showed a Mr for the enzyme of 61 kDa. The enzyme is not glycosylated. Structural 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 endoproteinase Lys C revealed six peptide fragments with 6-24 amino acids which were sequenced. The two largest fragments showed sequences, of which the motif 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% identity). Raucaffricine-O-beta-D-glucosidase seems to be a new member of the family 1 of glycosyl hydrolases.

Strictosidine synthase from Rauvolfia serpentina: analysis of a gene involved in indole alkaloid biosynthesis.

The gene for strictosidine synthase (str1), the enzyme which catalyzes the stereospecific condensation of tryptamine and secologanin to form the key indole alkaloid 3 alpha(S)-strictosidine has been isolated from genomic libraries prepared from Rauvolfia serpentina (India) and from Rauvolfia mannii (West Africa). The gene, str1, contained no introns and showed 100% nucleotide sequence homology over 1180 bp, encompassing the entire reading frame, between the two species. Transcription of the R. serpentina gene was found to start 81 nucleotides upstream from the AUG (26 nucleotides downstream from the TATA box). Transient expression assays in Nicotiana plumbaginifolia protoplasts of the R. serpentina str1 5'-noncoding region fused to the beta-glucuronidase reporter gene revealed promoter activity equivalent to 4 +/- 2% of that of 35 S CaMV promoter control. A series of truncated segments of the str1 promoter region indicated the presence of three areas of slight, but reproducible, negative control. Gel retardation assays demonstrated that several regions of the 5'-flanking sequences specifically bound nuclear protein from R. serpentina and that at least one region does not bind R. mannii nuclear protein. A survey of the expression of str1 in the R. serpentina plant suggested that strictosidine synthase poly(A)+ RNA was present predominantly, but not exclusively, in the root. This result correlated well with the distribution of both enzyme activity and indole alkaloids which were also predominant in the root, but, in general, distributed throughout the shrub.