Biosynthesis Of Ajmaline Research Articles

De novo biosynthesis of antiarrhythmic alkaloid ajmaline

The antiarrhythmic drug ajmaline is a monoterpenoid indole alkaloid (MIA) isolated from the Ayurvedic plant Rauvolfia serpentina (Indian Snakeroot). Research into the biosynthesis of ajmaline and another renowned MIA chemotherapeutic drug vinblastine has yielded pivotal advancements in the fields of plant specialized metabolism and engineering over recent decades. While the majority of vinblastine biosynthesis has been recently elucidated, the quest for comprehending ajmaline biosynthesis remains incomplete, marked by the absence of two critical enzymes. Here, we show the discovery and characterization of these two elusive reductases, alongside the identification of two physiologically relevant esterases that complete the biosynthesis of ajmaline. We show that ajmaline biosynthesis proceeds with vomilenine 1,2(R)-reduction followed by its 19,20(S)-reduction. This process is further modulated by two root-expressing esterases that deacetylate 17-O-acetylnorajmaline. Expanding upon the successful completion of the ajmaline biosynthetic pathway, we engineer the de novo biosynthesis of ajmaline in Baker’s yeast.

In silico characterization of Ajmaline biosynthesis pathway genes in plants

Alkaloids sourced from medicinally important plants have wide impact on human health and well-being. Ajmaline, a monoterpenoid indole alkaloid have divergent pharmacological applications and require large scale production. Rauvolfia serpentina (Apocynaceae) is an important source of ajmaline. Our present work encompasses computational phylogenetic approach towards understanding the ancestry and divergence of six of the most important enzymes of ajmaline biosynthetic pathway- Strictosidine synthase (STR1, EC 4.3.3.2), Strictosidine glucosidase (SGR1, EC 3.2.1.105), Polyneuridine aldehyde esterase (PNAE, EC 3.1.1.78), Vinorine synthase (ACT/VS, EC 2.3.1.160), Vinorine hydroxylase (CYP5437/VH, EC 1.14.13.75) and Acetylajmalan esterase (AAE, EC 3.1.1.80). The highly restricted distribution of the genes represents their conservative nature and other than the members of Apocynaceae the genes of Ajmaline biosynthesis were found to be distributed in Rubiaceae, Malvaceae, Solanaceae, Oleaceae, Gelsemiaceae, Fabaceae, Fragaceae etc. Identification of conserved domains of the enzymes were analysed based on the sequence homology of related plants. Amino acid sequences of STR1 showed highly variable N-terminal region in contrast to SGR1 and VH where the N-terminal sequence showed more conservation than C-terminal half. PNAE and VS showed high conservation throughout the sequence. Homology modeling and in silico structural analysis of the enzymes provide valuable insight for further structural and functional experimentation. The results reflected conservation of the core catalytic domain in all the six enzymes studied which is useful for structure-based evolution studies or for rational design and modulation of the enzyme's substrate specificity. Extensive literature survey resulted in identification of homologous genes of the aforementioned enzymes in Arabidopsis thaliana whose expressions at different stress conditions and developmental stages were investigated in publicly available microarray-based platform. The genes were found to be upregulated under different biotic and abiotic stresses. Also differential expression was detected at developmental stages.The data presented here will positively contribute towards deciphering the intricacies of reaction mechanism and steer bioengineering of alkaloid production to benefit mankind.

Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R.serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R.serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R.serpentina.

Sarpagan-Ajmalan-Type Indoles: Biosynthesis, Structural Biology, and Chemo-Enzymatic Significance.

The biosynthetic pathway of the monoterpenoid indole alkaloid ajmaline in the genus Rauvolfia, in particular Rauvolfia serpentina Benth. ex Kurz, is one of the few pathways that have been comprehensively uncovered. Every step in the progress of plant alkaloid biosynthesis research is due to the endeavors of several generations of scientists and the advancement of technologies. The tissue and cell suspension cultures developed in the 1970s by M.H. Zenk enabled the extraction of alkaloids and crude enzymes for use as experimental materials, thus establishing the foundation for further research on enzymatic reaction networks. In vivo NMR technology was first used in biosynthetic investigations in the 1990s following the invention of high-field cryo-NMR, which allowed the rapid and reliable detection of bioconversion processes within living plant cells. Shortly before, in 1988, a milestone was reached with the heterologous expression of the strictosidine synthase cDNA, which paved the way for the application of "reverse genetics" and "macromolecular crystallography." Both methods allowed the structural analysis of several Rauvolfia enzymes involved in ajmaline biosynthesis and expanded our knowledge of the enzyme mechanisms, substrate specificities, and structure-activity relationships. It also opened the door for rational enzyme engineering and metabolic steering. Today, the research focus of ajmaline biosynthesis is shifting from "delineation" to "utilization." The Pictet-Spenglerase strictosidine synthase, strictosidine glucosidase, together with raucaffricine glucosidase, as pioneers in this area, have become useful tools to generate "privileged structures" and "diversity oriented" syntheses, which may help to construct novel scaffolds and to set up libraries of sarpagan-ajmalan-type alkaloids in chemo-enzymatic approaches.

The molecular architecture of major enzymes from ajmaline biosynthetic pathway

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)8 barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.

Alkaloid Biosynthesis in Rauvolfia- cDNA Cloning of Major Enzymes of the Ajmaline Pathway

All the major reactions taking part in the biosynthesis of ajmaline in cell suspension cultures of the Indian medicinal plant Rauvolfia have now been established at the enzyme level. Of the well investigated 10 steps six of the involved enzymes have been functionally cloned. These are strictosidine synthase (STR), strictosidine glucosidase (SG), polyneuridine aldehyde esterase (PNAE), vinorine synthase (VS), cytochrome P450 reductase (CPR) and acetylajmalan acetylesterase (AAE). Because the cDNAs of these enzymes are now known their detailed molecular analysis became attainable for the first time. Some of these enzymes such as STR, SG or VS could be produced in E. coli at a preparative scale resulting in mg amounts of pure enzymes. They also have been crystallized and their preliminary X-ray analyses were published recently. It is only a matter of time that their molecular structure and the mechanisms of the catalyzed reactions will be elucidated. Of the soluble enzymes vomilenine reductase and 1.2-dihydrovomilenine reductase remain to be heterologously expressed. Appropriate cDNA clones have recently been isolated. What membrane bound enzymes of this pathway is concerned cloning could not be achieved up to now. Our future strategy is to purify these enzymes first and to use the

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

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 M r for the enzyme of ∼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.

In vivo monitoring of alkaloid metabolism in hybrid plant cell cultures by 2D cryo-NMR without labelling

Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic hybrid from Rauvolfia serpentina Benth. ex Kurz and Rhazya stricta Decaisne were carried out. When cell samples were taken sequentially from a stock feeding experiment, measuring times for in vivo NMR of 40 min were sufficient for following conversions of alkaloids at the natural abundance of 13C. Degradation of ajmaline added to the cells at 1.6 mM concentration to raumacline could be monitored after 96 h on a standard 800 MHz NMR instrument (Avance 800). Feeding vinorine an intermediate of ajmaline biosynthesis at 1.8 mM showed with a 500 MHz CryoProbe™ that the alkaloid enters two metabolic routes. Vinorine is intracellularly transformed on route I through vellosimine and 10-deoxysarpagine into sarpagine. On route II, the alkaloid is converted by hydroxylation through vomilenine into the glucoside raucaffricine. Intracellular alkaloid concentrations of ∼500 μM are measurable in vivo with cryogenic NMR technology.

A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline.

A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

Vomilenine Reductase — a novel Enzyme catalyzing a crucial Step in the Biosynthesis of the Therapeutically applied Antiarrhythmic Alkaloid Ajmaline

Delineation of the biochemical pathway leading to the antiarrhythmic Rauvolfia alkaloid ajmaline has been an important target in biosynthetic research for many years. The biosynthetic sequence starting with tryptamine and the monoterpene secologanin consists of about 10 different steps. Most of the participating enzymes have been detected and characterized previously, except those catalyzing the reduction of the intermediate vomilenine. A novel NADPH-dependent enzyme that reduces the intermediate has been isolated from Rauvolfia serpentina cell suspension cultures. Vomilenine reductase (M r 43 kDa, temp opt 30 °C, pH opt 5.7–6.2), saturates the indolenine double bond of vomilenine with stereospecific formation of 2β( R)-1,2-dihydrovomilenine. The described detection, enrichment and properties of the reductase not only closes a gap in ajmaline biosynthesis but is also a prerequisite for overexpressing the protein heterologously for final clarification of its molecular properties.

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-β- 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 ( K M 1.3 mM, V max 0.5 nkat/μg 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 ( K M 1.8 mM, V max 2.6 pkat/μg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.

Enzymatic Biosynthesis of Vomilenine, a Key Intermediate of the Ajmaline Pathway, Catalyzed by a Novel Cytochrome P 450-Dependent Enzyme from Plant Cell Cultures of Rauwolfia serpentina

Abstract Microsomal preparations from Rauwolfia serpentina Benth. cell suspension cultures cata­lyze a key step in the biosynthesis of ajmaline -the enzymatic hydroxylation of the indole alkaloid vinorine at the allylic C-21 resulting in vomilenine. Vomilenine is an important branch-point intermediate, leading not only to ajmaline but also to several side reactions of the biosynthetic pathway to ajmaline. The investigation of the taxonomical distribution of the enzyme indicated that vinorine hydroxylase is exclusively present in ajmaline-producing plant cells. The novel enzyme is strictly dependent on NADPH2 and O2 and can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole and carbon mon­oxide (the effect of CO is reversible with light of 450 nm). This suggests that vinorine hy­droxylase is a cytochrome P450-dependent monooxygenase. A pH optimum of 8.3 and a temperature optimum of 40 °C were found. The Km value was 3 μᴍ for NADPH2 and 26 μᴍ for vinorine. The optimum enzyme activity could be determined at day 4 after inoculation of the cell cultures in AP I medium. Vinorine hydroxylase could be stored with 20% sucrose at -28 °C without significant loss of activity.

RLCC-isolation of Raucaffricine from its most efficient source ? cell suspension cultures of Rauwolfia serpentina Benth

Raucaffricine (vomilenine-galactoside) was shown to be the major indole alkaloid of Rauwolfia serpentina Benth. cell suspension cultures grown in AP-medium (alkaloid production medium). Several grams of this glycoalkaloid can conveniently be isolated by RLCC (Rotation Locular Countercurrent Chromatography). A newly discovered enzyme efficiently converts the glycoalkaloid to its aglycon, vomilenine, which occupies a key function in the biosynthesis of ajmaline. This is the first demonstration of the occurrence of raucaffricine in Rauwolfia serpentina.