Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaloidal network of the plant Rauvolfia.

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a "reverse-genetic" approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His(6)-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.

Purification and partial amino acid sequences of the enzyme vinorine synthase involved in a crucial step of ajmaline biosynthesis.

The acetyl-CoA-dependent enzyme vinorine synthase was isolated from hybrid cell suspension cultures of Rauvolfia serpentina and Rhazya stricta. The sarpagan-type alkaloid gardneral was used as a substrate of the enzyme leading to the ajmalan-type 10-methoxyvinorine. An HPLC-based assay was developed to monitor vinorine synthase activity, which allowed establishing a five step purification procedure combining anion exchange, hydrophobic interaction, hydroxyapatite and gel filtration. Purification resulted in a yield of 0.2% and an approximately 991-fold enrichment of the acetyltransfer activity. SDS-PAGE analysis showed a Mr for the enzyme of approximately 50 kDa. The four peptide fragments generated by proteolysis of the pure enzyme with endoproteinase LysC and the N-terminal part of the enzyme were sequenced. The enzyme preparation (> 875-fold enrichment) delivering the N-terminal sequence was isolated from R. serpentina cell suspensions. Sequence alignment of the five peptides showed highest homologies in a range of 30-71% to acetyltransferases from other higher plants involved in natural plant product biosynthesis. Based on the partial sequences vinorine synthase is probably a novel member of the BAHD enzyme super family.

New alkaloids of the sarpagine group from Rauvolfia serpentina hairy root culture.

Three new monoterpenoid indole alkaloids, 19(S),20(R)-dihydroperaksine (1), 19(S),20(R)-dihydroperaksine-17-al (2), and 10-hydroxy-19(S),20(R)-dihydroperaksine (3), along with 16 known alkaloids 4-19 were isolated from hairy root culture of Rauvolfia serpentina, and their structures were elucidated by 1D and 2D NMR analyses. Taking into account the stereochemistry of the new alkaloids and results of preliminary enzymatical studies, the putative biosynthetical relationships between the novel alkaloids are discussed.

Isolation and structure elucidation of a new indole alkaloid from Rauvolfia serpentina hairy root culture: the first naturally occurring alkaloid of the raumacline group.

A new monoterpenoid indole alkaloid, 10-hydroxy- N(alpha)-demethyl-19,20-dehydroraumacline ( 1), was isolated as a mixture of E- and Z-isomers from hairy root culture of Rauvolfia serpentina Benth. ex Kurz (Apocynaceae) and the structure was determined by 1D and 2D NMR analyses. The new indole alkaloid represents the first naturally occurring alkaloid of the raumacline group and its putative biosynthetical pathway is discussed.

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids.

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (Nbeta-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation.