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1.
Plant J ; 87(4): 335-42, 2016 08.
Article in English | MEDLINE | ID: mdl-27122470

ABSTRACT

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 30 years. 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 Nß -methylajmaline, an unusual charged MIA found in R. serpentina.


Subject(s)
Ajmaline/biosynthesis , Methyltransferases/metabolism , Rauwolfia/enzymology , Secologanin Tryptamine Alkaloids/metabolism , Ajmaline/chemistry , Biosynthetic Pathways , Cloning, Molecular , Computational Biology , Methyltransferases/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/chemistry , Plant Roots/enzymology , Plant Roots/genetics , Rauwolfia/chemistry , Rauwolfia/genetics , Recombinant Proteins , Secologanin Tryptamine Alkaloids/chemistry , Substrate Specificity
2.
Alkaloids Chem Biol ; 76: 1-61, 2016.
Article in English | MEDLINE | ID: mdl-26827882

ABSTRACT

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.


Subject(s)
Ajmaline/biosynthesis , Indole Alkaloids/metabolism , Ajmaline/chemistry , Glucosidases/metabolism , Indole Alkaloids/chemistry , Magnetic Resonance Spectroscopy , Secologanin Tryptamine Alkaloids/chemistry , Secologanin Tryptamine Alkaloids/metabolism
3.
Alkaloids Chem Biol ; 76: 63-169, 2016.
Article in English | MEDLINE | ID: mdl-26827883

ABSTRACT

The sarpagine-related macroline and ajmaline alkaloids share a common biosynthetic origin, and bear important structural similarities, as expected. These indole alkaloids are widely dispersed in 25 plant genera, principally in the family Apocynaceae. Very diverse and interesting biological properties have been reported for this group of natural products. Isolation of new sarpagine-related alkaloids and the asymmetric synthesis of these structurally complex molecules are of paramount importance to the synthetic and medicinal chemists. A total of 115 newly isolated sarpagine-related macroline and ajmaline alkaloids, along with their physicochemical properties have been included in this chapter. A general and efficient strategy for the synthesis of these monomeric alkaloids, as well as bisindoles, has been presented, which involves application of the asymmetric Pictet-Spengler reaction (>98% ee) as a key step because of the ease of scale up of the tetracyclic template. Also included in this chapter are the syntheses of the sarpagine-related alkaloids, published since 2000.


Subject(s)
Indole Alkaloids/metabolism , Secologanin Tryptamine Alkaloids/metabolism , Ajmaline/biosynthesis , Ajmaline/chemistry , Indole Alkaloids/chemistry , Magnetic Resonance Spectroscopy , Secologanin Tryptamine Alkaloids/chemistry
4.
Planta ; 222(5): 888-98, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16133216

ABSTRACT

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.


Subject(s)
Ajmaline/biosynthesis , Esterases/metabolism , Rauwolfia/metabolism , Ajmaline/chemistry , Amino Acid Sequence , Base Sequence , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Cloning, Molecular , DNA, Complementary/genetics , DNA, Plant/genetics , Esterases/genetics , Gene Expression , Genes, Plant , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinetics , Molecular Sequence Data , Plants, Genetically Modified , Rauwolfia/genetics , Rauwolfia/virology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Nicotiana/enzymology , Nicotiana/genetics
5.
J Biol Chem ; 280(14): 13576-83, 2005 Apr 08.
Article in English | MEDLINE | ID: mdl-15665331

ABSTRACT

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.


Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Structure, Tertiary , Acetyltransferases/genetics , Ajmaline/biosynthesis , Amino Acid Sequence , Anti-Arrhythmia Agents/metabolism , Coenzyme A/chemistry , Coenzyme A/metabolism , Crystallography, X-Ray , Indole Alkaloids/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Plant Proteins/genetics , Sequence Alignment
6.
Bioorg Med Chem ; 12(10): 2781-6, 2004 May 15.
Article in English | MEDLINE | ID: mdl-15110859

ABSTRACT

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.


Subject(s)
Acetyl-CoA C-Acetyltransferase/chemistry , Acetyl-CoA C-Acetyltransferase/isolation & purification , Ajmaline/biosynthesis , Acetyl-CoA C-Acetyltransferase/metabolism , Amino Acid Sequence , Apocynaceae/enzymology , Hybrid Cells/enzymology , Indole Alkaloids , Molecular Sequence Data , Molecular Structure , Rauwolfia/enzymology , Sequence Analysis, Protein
7.
Biotechnol Lett ; 25(8): 631-6, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12882157

ABSTRACT

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2-3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l-1 and 0.1 mg alpha-naphthaleneacetic acid l-1 produced more ajmaline (0.01 mg g-1 dry wt) and ajmalicine (0.006 mg g-1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g-1 dry wt) and ajmalicine (0.0007 mg g-1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.


Subject(s)
Ajmaline/biosynthesis , Plant Roots/metabolism , Rauwolfia/metabolism , Secologanin Tryptamine Alkaloids , Yohimbine/analogs & derivatives , Yohimbine/metabolism , Cell Differentiation , Culture Techniques/methods , Indoleacetic Acids/pharmacology , Plant Roots/cytology , Plant Roots/growth & development , Plant Roots/microbiology , Plants, Medicinal/cytology , Plants, Medicinal/growth & development , Plants, Medicinal/metabolism , Plants, Medicinal/microbiology , Rauwolfia/cytology , Rauwolfia/growth & development , Rauwolfia/microbiology , Rhizobium/physiology
8.
Planta Med ; 68(10): 906-11, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12391554

ABSTRACT

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.


Subject(s)
Ajmaline/biosynthesis , Anti-Arrhythmia Agents/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/chemistry , Phytotherapy , Rauwolfia , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Humans
9.
Bioorg Med Chem ; 10(6): 1913-8, 2002 Jun.
Article in English | MEDLINE | ID: mdl-11937349

ABSTRACT

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 degrees C, pH opt 5.7-6.2), saturates the indolenine double bond of vomilenine with stereospecific formation of 2beta(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.


Subject(s)
Ajmaline/biosynthesis , Anti-Arrhythmia Agents/metabolism , Indole Alkaloids , Oxidoreductases/isolation & purification , Oxidoreductases/metabolism , Rauwolfia/enzymology , Secologanin Tryptamine Alkaloids/metabolism , Ajmaline/chemistry , Anti-Arrhythmia Agents/chemistry , Catalysis , Cells, Cultured , Chromatography , Hydrogen-Ion Concentration , Molecular Structure , NADP/metabolism , Oxidoreductases/chemistry , Rauwolfia/metabolism , Secologanin Tryptamine Alkaloids/chemistry , Temperature
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