Anthranilate N-methyltransferase, a branch-point enzyme of acridone biosynthesis

Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N-methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli, and shown to encode S-adenosyl-l-methionine-dependent anthranilate N-methyltransferase. SDS-PAGE of the purified enzyme revealed a mass of 40 +/- 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O-methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity ( approximately 20%) to N-methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O-methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3- and 4-aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N-methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT-PCR amplifications and Western blotting revealed expression of the N-methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.     

Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens.

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl-CoAs with N-methylanthraniloyl-CoA or 4-coumaroyl-CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4-Coumaroyl-CoA was the preferred starter substrate for CHS1, but cinnamoyl-CoA and caffeoyl-CoA were also turned over at significant rates. However, N-methylanthraniloyl-CoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4-coumaroyl-CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with N-methylanthraniloyl-CoA as the starter substrate, while the conversion of cinnamoyl-CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site-directed mutagenesis.     

Native acridone synthases I and II from Ruta graveolens L. form homodimers

Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L. and expressed in Escherichia coli. The translated polypeptide of Mr 42,681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70-75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells. Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four-step purification protocol, and the affinities to N-methylanthraniloyl-CoA and malonyl-CoA were determined. The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44 +/- 3 kDa found for the acridone synthase I on Superdex 75. Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81 +/- 4 kDa for both acridone synthases. It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.     

Transformation of acridone synthase to chalcone synthase.

Acridone synthase (ACS) and chalcone synthase (CHS) catalyse the pivotal reactions in the formation of acridone alkaloids or flavonoids. While acridone alkaloids are confined almost exclusively to the Rutaceae, flavonoids occur abundantly in all seed-bearing plants. ACSs and CHSs had been cloned from Ruta graveolens and shown to be closely related polyketide synthases which use N-methylanthraniloyl-CoA and 4-coumaroyl-CoA, respectively, as the starter substrate to produce the acridone or naringenin chalcone. As proposed for the related 2-pyrone synthase from Gerbera, the differential substrate specificities of ACS and CHS might be attributed to the relative volume of the active site cavities. The primary sequences as well as the immunological cross reactivities and molecular modeling studies suggested an almost identical spatial structure for ACS and CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133 and Val265 were assumed to play a critical role in substrate specificity. Exchange of a single amino acid (Val265Phe) reduced the catalytic activity by about 75% but grossly shifted the specificity towards CHS activity, and site-directed mutagenesis replacing all three residues by the corresponding amino acids present in CHS (Ser132Thr, Ala133Ser and Val265Phe) fully transformed the enzyme to a functional CHS with comparatively marginal ACS activity. The results suggested that ACS divergently has evolved from CHS by very few amino acid exchanges, and it remains to be established why this route of functional diversity has developed in the Rutaceae only.     

Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae

Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.     

Potential anti-allergic acridone alkaloids from the roots of Atalantia monophylla

Acridone alkaloids, cycloatalaphylline-A (1), N-methylcyclo-atalaphylline-A (2) and N-methylbuxifoliadine-E (3), were isolated from the dichloromethane and acetone extracts of the root of Atalantia monophylla along with eight known acridone alkaloids: buxifoliadine-A (4), buxifoliadine-E (5), N-methylatalaphylline (6), atalaphylline (7), citrusinine-I (8), N-methylataphyllinine (9), yukocitrine (10) and junosine (11) and two known coumarins: auraptene (12) and 7-O-geranylscopoletin (13). Their structures were elucidated on the basis of spectroscopic analyses. Compounds 2, 5 and 8 possessed appreciable anti-allergic activity in RBL-2H3 cells model with IC(50) values of 40.1, 6.1 and 18.7 microM, respectively.     

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).     

Anthranilate synthase from Ruta graveolens. Duplicated AS alpha genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.

Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the biosynthetic precursor of both tryptophan and numerous secondary metabolites, including inducible plant defense compounds. The higher plant Ruta graveolens produces tryptophan and elicitor-inducible, anthranilate-derived alkaloids by means of two differentially expressed nuclear genes for chloroplast-localized AS alpha subunits, AS alpha 1 and AS alpha 2. Mechanisms that partition chorismate between tryptophan and inducible alkaloids thus do not entail chloroplast/cytosol separation of AS isoenzymes and yet might involve differential feedback regulation of pathway-specific AS alpha subunits. The two AS alpha isoenzymes of R. graveolens were expressed as glutathione S-transferase fusion proteins in Escherichia coli deletion mutants defective in AS activity and were purified to homogeneity. Differential sensitivity of the transformed E. coli strains toward 5-methyltryptophan, a false-feedback inhibitor of AS, was demonstrated. Characterization of affinity-purified AS alpha isoenzymes revealed that the noninducible AS alpha 2 of R. graveolens is strongly feedback inhibited by 10 microns tryptophan. In contrast, the elicitor-inducible AS alpha 1 isoenzyme is only slightly affected even by tryptophan concentrations 10-fold higher than those observed in planta. These results are consistent with the hypothesis that chorismate flux into biosynthesis of tryptophan and defense-related alkaloid biosynthesis in R. graveolens is regulated at the site of AS alpha isoenzymes at both genetic and enzymatic levels.     

N-Substituted acridone alkaloids from Toddaliopsis bremekampii (Rutaceae: Toddalioideae) of south-central Africa

Toddaliopsins A-D, four novel 1,2,3-trioxygenated acridone alkaloids, have been isolated from the leaves of Toddaliopsis bremekampii. Toddaliopsins B-D are the first reported acridone alkaloids with substituted N-methyl groups, in the light of which the chemotaxonomic relationship of Toddaliopsis and Vepris is discussed. Toddaliopsin C possesses moderate anti-inflammatory activity, which may be related to the hydroxy group present at C-1.     

Seasonal variation of chemical profile of Ruta graveolens extracts and biological activity against Fusarium oxysporum,Fusarium proliferatum and Stemphylium vesicarium

Ruta graveolens L. is a plant of agricultural interest due to its biological activity against phytopathogenic microorganism. This activity has been attributed to the presence of alkaloids, furanocoumarins, terpenes and fatty acids. The accumulation of these compounds is affected by environmental factors. The aim of this work was to analyze the seasonal variation the chemical profile of rue and to evaluate the biological activity against phytopathogenic fungi. Extracts were obtained in January, April, June and September 2017, chemically characterized by GC-MS. The antifungal activity was determined by absorbance, four concentrations were evaluated: 4, 8, 16 and 32 mg/mL. In extracts from the aerial parts, fatty acids were the most abundant group in the January sample (29.7%), and alkaloids in the April and June (27.7%) samples. In extracts from the roots, alkaloids were the most abundant group in the four collections. The highest percentage of growth inhibition on Fusarium and Stemphylium was obtained with the extracts of the January sample. Ruta graveolens can be considered an alternative for the management of phytopathogenic fungi of crops of economic importance.     

Acridone and furoquinoline alkaloids from Teclea gerrardii (Rutaceae: Toddalioideae) of southern Africa

The combined hexane/CH(2)Cl(2) extract of the stem bark of Teclea gerrardii (Rutaceae: Toddalioideae) has yielded two acridone alkaloids, 3-hydroxy-1-methoxy-N-methylacridone (tegerrardin A) (1) and 3-hydroxy-N-methyl-1-(gamma,gamma-dimethylallyloxy)acridone (tegerrardin B) (2), three known acridones (3-5), two known furoquinolines (6,7), and the acridone precursor tecleanone (8). Arborinine (3) and evoxine (6) displayed moderate antiplasmodial activity against the CQS D10 strain of Plasmodium falciparum, with IC(50) values of 12.3 and 24.5 microM, respectively.     

Algicidal and antifungal compounds from the roots of Ruta graveolens and synthesis of their analogs

Bioassay-guided fractionation of the ethyl acetate extract of Ruta graveolens roots yielded rutacridone epoxide with potent selective algicidal activity towards the 2-methyl-isoborneol (MIB)-producing blue-green alga Oscillatoria perornata, with relatively little effect on the green alga Selenastrum capricornutum. The diol-analog of rutacridone epoxide, gravacridondiol, which was also present in the same extract, had significantly less activity towards O. perornata. Rutacridone epoxide also showed significantly higher activity than commercial fungicides captan and benomyl in our micro-bioassay against the agriculturally important pathogenic fungi Colletotrichum fragariae, C. gloeosporioides, C. acutatum, and Botrytis cineara and Fusarium oxysporium. Rutacridone epoxide is reported as a direct-acting mutagen, precluding its use as an agrochemical. In order to understand the structure-activity relationships and to develop new potential biocides without toxicity and mutagenicity, some analogs containing the (2-methyloxiranyl)-dihydrobenzofuran moiety with an epoxide were synthesized and tested. None of the synthetic analogs showed comparable activities to rutacridone epoxide. The absolute stereochemistry of rutacridone was determined to be 2'(R) and that of rutacridone epoxide to be 2'(R), 3'(R) by CD and NMR analysis.     

Mutagenic compounds in an extract from Rutae Herba (Ruta graveolens L.). II. UV-A mediated mutagenicity in the green alga Chlamydomonas reinhardtii by furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba

A commercial tincture prepared from Rutae Herba (Ruta graveolens L.) exhibited a moderate photomutagenicity in an arginine-requiring mutant strain of Chlamydomonas reinhardtii. In the tincture some furocoumarins, e.g., bergapten, psoralen, imperatorin, and 3 furoquinoline alkaloids (dictamnine, gamma-fagarine, skimmianine) were detected. All compounds revealed photomutagenic properties but their activities were quite different. Bergapten was the most potent furocoumarin. Dictamnine, the furoquinoline with the strongest effect, reached only about 10% of the activity of bergapten. Based on the amount of these compounds in the tincture and their activities we conclude that bergapten is mainly responsible for the photomutagenicity of the tincture. The lower phototoxicity and photomutagenicity of the furoquinoline alkaloids may be due to the fact that furoquinolines form only monoadducts with DNA in the presence of UV-A in contrast to furocoumarins which also form biadducts.     

Potential allelochemicals from the essential oil of Ruta graveolens

The essential oil of aerial parts of Ruta graveolens was obtained by hydrodistillation with a 0.74% yield on a dry weight basis. Thirty-eight components were identified by GC and GC-MS analyses. 2-Ketones predominated in the essential oil, with undecan-2-one (46.8%) and nonan-2-one (18.8%) as the main constituents. The essential oil and some of its constituents were tested for their allelopathic activity in vitro on radish germination and radicle growth in light and darkness. The essential oil and some of its minor constituents were effective and dose-dependent inhibitors of both the germination and radicle growth; 2-ketones are not active. The possible allelopathic activity of rue essential oil and some its isolated constituents is reported.     

Evaluation and lead optimization of anti-malarial acridones

With 2-methoxy-6-chloroacridone as a lead compound, we synthesized and tested acridone derivatives to develop a better understanding of the anti-malarial structure-activity relationships. Over 30 acridone derivatives were synthesized. The most potent compounds contained extended alkyl chains terminated by trifluoromethyl groups and located at the 3-position of the tricyclic system. Acridones optimized in the length of the side chain and the nature of the terminal fluorinated moiety exhibited in vitro anti-malarial IC(50) values in the low nanomolar and picomolar range and were without cytotoxic effects on the proliferation and differentiation of human bone marrow progenitors or mitogen-activated murine lymphocytes at concentrations up to 100,000-fold higher. Based on a structural similarity to known anti-malarial agents it is proposed that the haloalkoxyacridones exert their anti-malarial effects through inhibition of the Plasmodium cytochrome bc(1) complex. Haloalkoxyacridones represent an extraordinarily potent novel class of chemical compounds with the potential for development as therapeutic agents to treat or prevent malaria in humans.     

芸香地上器官的生物学研究

对芸香的茎、叶及花等器官的形态结构进行了较为系统的观察研究,对花芽形成及开花过程进行了客观描述.芸香花有顶生和侧生两种,柱头为"湿柱头",叶片具有典型旱生型植物特点,茎叶中均含有簇晶和油腔,这些特征可作为分类学上鉴别该物种的性状.茎的淀粉积累过程:花前期淀粉积累很少(6月下旬),10月下旬达高峰,之后略有减少并保持稳定直至被冻死.对此结合当地温度及水分条件进行了抗寒、抗旱的讨论.     

Polar constituents of celery seed

From the water-soluble portion of the methanol extract of celery seed (fruit of Apium graveolens L.) five sesquiterpenoid glucosides (celerioside A-E) and three phthalide glycosides (celephtalide A-C) were isolated together with six aromatic compound glucosides, two norcarotenoid glucosides and a lignan glucoside. Their structures were determined by spectral investigations.     

Antileishmanial and antifungal acridone derivatives from the roots of Thamnosma rhodesica

Eight furanocoumarins, one coumarin and four acridone derivatives have been identified in the roots of Thamnosma rhodesica (Rutaceae). Rhodesiacridone, one of these acridone derivatives, is reported here for the first time. Its structure was elucidated by spectrometric methods including ESI-HR, EI, DCI mass spectrometry, 1H, 13C and 2D NMR experiments. This novel compound showed activities against the intracellular form of a human pathogen, the protozoan parasite Leishmania major. Two known acridone related compounds, gravacridonediol and 1-hydroxy-10-methylacridone, exhibited activities against the intracellular form of the same parasite and the fungus Cladosporium cucumerinum, respectively.     

Furoquinoline alkaloids from Teclea nobilis

Five new furoquinoline alkaloids, namely tecleabine (1), tecleoxine (2), isotecleoxine (3), methylnkolbisine (4) and chlorodesnkolbisine (5) were isolated from the aerial parts of Teclea nobilis, together with seven known furoquinoline derivatives; one acridone alkaloid, and one known flavanone. The structures of the alkaloids 1-5 were established by 1D and 2D NMR spectral data, including COSY, HMQC and HMBC experiments, as well as HRMS.