Publications

• 3D-Structure and function of strictosidine synthase--the key enzyme of monoterpenoid indole alkaloid biosynthesis.

  Stockigt J., Barleben L., Panjikar S., Loris E.A.

  Strictosidine synthase (STR; EC 4.3.3.2) plays a key role in the biosynthesis of monoterpenoid indole alkaloids by catalyzing the Pictet-Spengler reaction between tryptamine and secologanin, leading exclusively to 3alpha-(S)-strictosidine. The structure of the native enzyme from the Indian medicin ... >> More

  Strictosidine synthase (STR; EC 4.3.3.2) plays a key role in the biosynthesis of monoterpenoid indole alkaloids by catalyzing the Pictet-Spengler reaction between tryptamine and secologanin, leading exclusively to 3alpha-(S)-strictosidine. The structure of the native enzyme from the Indian medicinal plant Rauvolfia serpentina represents the first example of a six-bladed four-stranded beta-propeller fold from the plant kingdom. Moreover, the architecture of the enzyme-substrate and enzyme-product complexes reveals deep insight into the active centre and mechanism of the synthase highlighting the importance of Glu309 as the catalytic residue. The present review describes the 3D-structure and function of R. serpentina strictosidine synthase and provides a summary of the strictosidine synthase substrate specificity studies carried out in different organisms to date. Based on the enzyme-product complex, this paper goes on to describe a rational, structure-based redesign of the enzyme, which offers the opportunity to produce novel strictosidine derivatives which can be used to generate alkaloid libraries of the N-analogues heteroyohimbine type. Finally, alignment studies of functionally expressed strictosidine synthases are presented and the evolutionary aspects of sequence- and structure-related beta-propeller folds are discussed. << Less

  Plant Physiol Biochem 46:340-355(2008) [PubMed] [EuropePMC]

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

  Ruppert M., Woll J., Giritch A., Genady E., Ma X., Stockigt J.

  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 i ... >> More

  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. << Less

  Planta 222:888-898(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Substrate specificity of strictosidine synthase.

  McCoy E., Galan M.C., O'Connor S.E.

  Strictosidine synthase catalyzes a Pictet-Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids to generate strictosidine. The substrate requirements for strictosidine synthase are systematically and quantitatively examined and the enzymatically generated compounds ar ... >> More

  Strictosidine synthase catalyzes a Pictet-Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids to generate strictosidine. The substrate requirements for strictosidine synthase are systematically and quantitatively examined and the enzymatically generated compounds are processed by the second enzyme in this biosynthetic pathway. << Less

  Bioorg Med Chem Lett 16:2475-2478(2006) [PubMed] [EuropePMC]

• The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins.

  Ma X., Panjikar S., Koepke J., Loris E., Stockigt J.

  The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family represe ... >> More

  The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family. << Less

  Plant Cell 18:907-920(2006) [PubMed] [EuropePMC]

• Strictosidine: from alkaloid to enzyme to gene.

  Kutchan T.M.

  In this review, the elucidation of the structure of the first key alkaloidal intermediate in monoterpenoid indole alkaloid biosynthesis, 3 alpha(S)-strictosidine, is presented. The discovery of the enzyme which catalyses the stereospecific formation of this alkaloidal precursor from tryptamine and ... >> More

  In this review, the elucidation of the structure of the first key alkaloidal intermediate in monoterpenoid indole alkaloid biosynthesis, 3 alpha(S)-strictosidine, is presented. The discovery of the enzyme which catalyses the stereospecific formation of this alkaloidal precursor from tryptamine and secologanin, strictosidine synthase, is also detailed. From the knowledge provided by the stereochemical structure of strictosidine and the biochemical characteristics of the biosynthetic enzyme, strictosidine synthase, a new approach to the study of monoterpenoid indole alkaloid biosynthesis was developed. Physiological studies of monoterpenoid indole alkaloid biosynthesis at the enzymic level in plants and plant cell cultures were performed followed by the analyses of these systems at the level of molecular genetics. << Less

  Phytochemistry 32:493-506(1993) [PubMed] [EuropePMC]

• Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms.

  de Waal A., Meijer A.H., Verpoorte R.

  Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized. A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. Four of six isoforms were ... >> More

  Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized. A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. Four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous. All strictosidine synthase isoforms were found to be glycoproteins. The isoforms were also found in leaves and roots of the plant, in seedlings and in hairy root cultures. The ratio of the different isoforms differed slightly between these sources. The kinetic parameters of the isoforms showed no significant differences. The maximal velocity (300-400 nkat/mg of protein) is the highest reported so far. It was demonstrated that the apparent Michaelis constant for tryptamine (approx. 9 microM) is much lower than values reported previously. The presence of weak product inhibition (Kp approx. 35 times Km) was established, whereas substrate inhibition was not detected. << Less

  Biochem J 306:571-580(1995) [PubMed] [EuropePMC]