Strictosidine synthase, an indispensable enzyme involved in the biosynthesis of terpenoid indole and ß-carboline alkaloids.

Terpenoid indole (TIAs) and ß-carboline alkaloids (BCAs), such as suppressant reserpine, vasodilatory yohimbine, and antimalarial quinine, are natural compounds derived from strictosidine. These compounds can exert powerful pharmacological effects but be obtained from limited source in nature. the whole biosynthetic pathway of TIAs and BCAs, The Pictet-Spengler reaction catalyzed by strictosidine synthase (STR; EC: 4.3.3.2) is the rate-limiting step. Therefore, it is necessary to investigate their biosynthesis pathways, especially the role of STR, and related findings will support the biosynthetic generation of natural and unnatural compounds. This review summarizes the latest studies concerning the function of STR in TIA and BCA biosynthesis, and illustrates the compounds derived from strictosidine. The substrate specificity of STR based on its structure is also summarized. Proteins that contain six-bladed four-stranded ß-propeller folds in many organisms, other than plants, are listed. The presence of these folds may lead to similar functions among organisms. The expression of STR gene can greatly influence the production of many compounds. STR is mainly applied to product various valuable drugs in plant cell suspension culture and biosynthesis in other carriers.

Bacillus subtilis CBR05 induces Vitamin B6 biosynthesis in tomato through the de novo pathway in contributing disease resistance against Xanthomonas campestris pv. vesicatoria.

Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.

An atypical strictosidine synthase, OsSTRL2, plays key roles in anther development and pollen wall formation in rice.

Strictosidine synthase (STR) plays an important role in the biosynthesis of terpenoid indole alkaloids (TIAs) and is expressed in a range of active meristematic tissues of higher plants. STR proteins are involved in different physiological and biochemical pathways. However, the function of STR proteins in rice development remains poorly understood. In this study, we identified 21 possible STR-like (OsSTRL) family members in rice genome and found that only one gene, OsSTRL2, exhibited a pre-emergency specific florescence expression pattern. Tissue-specific expression profile analysis, ß-glucuronidase histochemical (GUS) staining and RNA in situ hybridization confirmed that OsSTRL2 was highly expressed in tapetal cells and microspores. Comparative protein sequence analysis indicated that OsSTRL2 lacked the key catalytic residue found in a typical STR (STR1), although it possessed conserved ß-propellers and α-helices formed the basic structure of STR1. OsSTRL2 knockout mutant resulted to male sterility because of the defects in anther development and pollen wall formation. Subcellular localization of OsSTRL2-YFP revealed that the OsSTRL2 protein was primarily localized in the endoplasmic reticulum (ER). Therefore, OsSTRL2 is an atypical strictosidine synthase that plays crucial roles in regulating anther development and pollen wall formation in rice.

Engineering strictosidine synthase: rational design of a small, focused circular permutation library of the ß-propeller fold enzyme.

Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of strictosidine analogs have however been of limited success due to the high substrate specificity of these enzymes. We have explored the impact of a protein engineering approach called circular permutation on the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the discovery process, our study departs from the usual process of creating a random protein library, followed by extensive screening. Instead, a small, focused library of circular permutated variants of the six bladed ß-propeller protein was prepared, specifically probing two regions which cover the enzyme active site. The observed activity changes suggest important roles of both regions in protein folding, stability and catalysis.

[Highly active fractions of the medicinal leech recombinant destabilase-lysozyme].

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

Construction and expression of a dual vector for chemo-enzymatic synthesis of plant indole alkaloids in Escherichia coli.

A dual vector (pQE-70-STR1-SG) containing coding regions of strictosidine synthase (STR1, EC 4.3.3.2) and strictosidine glucosidase (SG, EC 3.2.1.105) from the Indian medicinal plant Rauvolfia serpentina was constructed. Functional expression of the vector in Escherichia coli cells (M15 strain) was proven by isolation of prepurified enzyme extracts, which show both STR1 and SG activities. Incubation of the enzyme in the presence of tryptamine and secologanin delivered the indole alkaloid cathenamine, demonstrating functional co-expression of both STR1- and SG-cDNAs. Cathenamine reduction by sodium borohydride leading to tetrahydroalstonine revealed the chemo-enzymatic indole alkaloid synthesis.

Establishment of a soybean (Glycine max Merr. L) transposon-based mutagenesis repository.

Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes, most with unknown function. Therefore, a major research priority is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed approximately 70% disrupted known genes, with the majority matching sequences derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by insertional mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.

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

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.

Structure-based engineering of strictosidine synthase: auxiliary for alkaloid libraries.

The highly substrate-specific strictosidine synthase (EC 4.3.3.2) catalyzes the biological Pictet-Spengler condensation between tryptamine and secologanin, leading to the synthesis of about 2000 monoterpenoid indole alkaloids in higher plants. The crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine has been elucidated here, allowing the rational site-directed mutation of the active center of STR1 and resulting in modulation of its substrate acceptance. Here, we report on the rational redesign of STR1 by generation of a Val208Ala mutant, further describing the influence on substrate acceptance and the enzyme-catalyzed synthesis of 10-methyl- and 10-methoxystrictosidines. Based on the addition of strictosidine to a crude strictosidine glucosidase preparation from Catharanthus cells, a combined chemoenzymatic approach to generating large alkaloid libraries for future pharmacological screenings is presented.

Analysis of the Arabidopsis rsr4-1/pdx1-3 mutant reveals the critical function of the PDX1 protein family in metabolism, development, and vitamin B6 biosynthesis.

Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. It has been demonstrated to alleviate oxidative stress and in its phosphorylated form participates as a cofactor in >100 biochemical reactions. By means of a genetic approach, we have identified a novel mutant, rsr4-1 (for reduced sugar response), with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Cloning of the mutated gene revealed that rsr4-1 carries a point mutation in a member of the PDX1/SOR1/SNZ (for Pyridoxine biosynthesis protein 1/Singlet oxygen resistant 1/Snooze) family that leads to reduced vitamin B6 content. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers. Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants.

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

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.

Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses.

Pyridoxine (vitamin B6) is a cofactor required by numerous enzymes in all cellular organisms. Plants are the major source of vitamin B6 for animals, yet the biosynthesis pathway and the function of vitamin B6 in plants are not well elucidated. In this study, an Arabidopsis pyridoxine synthase gene PDX1 was characterized and its in vivo functions were investigated. The PDX1 gene was expressed in all plant parts examined and its expression level was not significantly regulated by abiotic stress or the phytohormone abscisic acid. In roots, PDX1 was highly expressed in a defined region behind the root tips that undergoes rapid cell division. The PDX1 protein was mainly associated with the plasma membrane and endomembranes, implying a potential involvement of vitamin B6 in membrane function. To reveal the in vivo role of PDX1, Arabidopsis insertional mutants were isolated. Strikingly, the pdx1 knockout mutants were impaired in root growth and early seedling development. The stunted roots resulted from both reduced cell division and elongation. Supplementation of the growth media with pyridoxine or reintroduction of the wild-type PDX1 gene into the mutants completely restored the mutant growth, demonstrating that PDX1 is required for pyridoxine biosynthesis in planta. In addition to the developmental defects, pdx1 mutants are hypersensitive to osmotic stress and oxidative stress. These mutant seedlings had increased peroxidation of membrane lipids following UV treatment. Our study establishes a critical role of vitamin B6 in plant development and stress tolerance and suggests that vitamin B6 may represent a new class of antioxidant in plants.

Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus.

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.

Optimization of selenium substructures as obtained from SHELXD.

Using the signal of naturally inbuilt or artificially introduced anomalous scatterers to derive initial phases in a macromolecular crystal structure determination has become routine in recent years. In the context of high-throughput crystallography in particular, MAD and SAD (multiple- and single-wavelength anomalous dispersion) methods are central tools. For both techniques, a crucial step is the determination of the substructure of anomalous scatterers; subsequent phasing procedures will profit from a substructure model that is as accurate as possible. The choice of the subset of the diffraction data to be used for the substructure determination has a strong influence on the quality of the substructure and can make the difference between success and failure. The accuracy of selenium substructures obtained using F(A) values or various anomalous differences truncated to different resolutions has been investigated by comparing the sites determined by SHELXD with the selenium positions in the refined models. Based on the analysis, some recommendations for obtaining accurate and precise substructures are derived.

Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds.

Camptothecin derivatives are clinically used anti-tumor compounds that biogenetically belong to a group of monoterpenoid indole alkaloids (TIA). We have already established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) that produces camptothecin. The present study describes the cloning and characterization of cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of TIA from hairy roots of O. pumila. We also isolated the cDNA coding for NADPH:cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR. The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.

Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants.

Camptothecin derivatives are clinically used anti-neoplastic alkaloids that biogenetically belong to monoterpenoid indole alkaloids. Camptothecin-related alkaloids from the methanol extracts of Ophiorrhiza pumila, Camptotheca acuminata and Nothapodytes foetida plants were profiled and identified using a reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization ion-trap mass spectrometry. A natural 10-glycosyloxy camptothecin, chaboside, was accumulated in tissues of O. pumila but not in C. acuminata and N. foetida. Anthraquinones regarded as phytoalexins were present in the extracts of hairy roots and calli but not in the differentiated plants of O. pumila. These findings demonstrated a remarkable difference in the constituents between the differentiated plants and the hairy roots or calli tissues. The activity of strictosidine synthase, a key enzyme of camptothecin biosynthesis, was detected in the protein extracts of stems and roots of O. pumila, being correlated with the pattern of strictosidine synthase mRNA expression.

Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.

Investigation of the coupling reaction of tetraacetylsecologanin with oxotryptamine and its derivative.

The coupling reaction of tetraacetylsecologanin with 2,3-dihydro-2-oxotryptamine and its N(b)-benzyl derivative was investigated. With the benzylated amine, the reaction was stopped at the tetracyclic ester level, and with the unsubstituted amine it was immediately followed by lactamization. In both cases, the products were formed with high stereoselectivity at C-3, but as an epimeric pair of 7R and 7S in a ratio of 1:3. The bulky benzyl substituent at N-4 directed the stereoselectivity at C-3 in favor of the S configuration. In the nonbenzylated compounds, the reversible coupling reaction is probably nonstereoselective, but in lactamization the 3R epimer is sterically favored and faster and gives the final lactam in this configuration. The formation of the spiro compounds may serve as a model reaction in the interpretation of the stereoselectivity of the coupling reaction of secologanin with tryptamine in the presence of strictosidine synthase.

Destabilase from the medicinal leech is a representative of a novel family of lysozymes.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a JA-independent signal transduction pathway.

Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress hormonejasmonate. In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. Here we show that an upstream region in the Str promoter confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses showed that the Str promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P. CrBPF-1 mRNA accumulated rapidly in elicitor-treated C. roseus suspension cells, whereas no induction was observed with jasmonate. Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor-responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.