Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei

Five clustered polyketide synthase (PKS) genes, rifA–rifE, involved in rifamycin (Rf) biosynthesis in Amycolatopsis mediterranei S699 have been cloned and sequenced (August, P.R. et al., 1998. Chem. Biol. 5, 69–79). The five multifunctional polypeptides constitute a type I modular PKS that contains ten modules, each responsible for a specific round of polyketide chain elongation. Sequence comparisons of the Rf PKS proteins with other prokaryotic modular PKSs elucidated the regions that have an important role in enzyme activity and specificity. The β-ketoacyl:acyl carrier protein synthase (KS) domains show the highest degree of similarity between themselves (86–90%) and to other PKSs (78–85%) among all the constituent domains. Both malonyl-coenzyme A (MCoA) and methylmalonyl-coenzyme A (mMCoA) are substrates for chain elongation steps carried out by the Rf PKS. Since acyltransferase (AT) domains of modular PKSs can distinguish between these two substrates, comparison of the sequence of all ten AT domains of the Rf PKS with those found in the erythromycin (Er) (Donadio, S. and Katz, L., 1992. Gene 111, 51–60) and rapamycin (Rp) (Haydock, S. et al., 1995. FEBS Lett. 374, 246–248) PKSs revealed that the AT domains in module 2 of RifA and module 9 of RifE are specific for MCoA, whereas the other eight modules specify mMCoA. Dehydration of the β-hydroxyacylthioester intermediates should occur during the reactions catalysed by module 4 of RifB and modules 9 and 10 of RifE, yet only the active site region of module 4 conforms closely to the dehydratase (DH) motifs in the Er and Rp PKSs. The DH domains of modules 9 and 10 diverge significantly from the consensus sequence defined by the Er and Rp PKSs, except for the active site His residues. Deletions in the DH active sites of module 1 in RifA and module 5 in RifB and in the N- and C-terminal regions of module 8 of RifD should inactivate these domains, and module 2 of RifA lacks a DH domain, all of which are consistent with the proposed biosynthesis of Rf. In contrast, module 6 of RifB and module 7 of RifC appear to contain intact DH domains even though DH activity is not apparently required in these modules. Module 2 of RifA lacks a β-ketoacyl:acyl carrier protein reductase (KR) domain and the one in module 3 has an apparently inactive NADPH binding motif, similar to one found in the Er PKS, while the other eight KR domains of the Rf PKS should be functional. These observations are consistent with biosynthetic predictions. All the acyl carrier protein (ACP) domains, while clearly functional, nevertheless have active site signature sequences distinctive from those of the Er and Rp PKSs. Module 2 of RifA has only the core domains (KS, AT and ACP). The starter unit ligase (SUL) and ACP domains present in the N-terminus of RifA direct the selection and loading of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), onto the PKS. AHBA is made by the products of several other genes in the Rf cluster through a variant of the shikimate pathway (August, P.R. et al., inter alia). RifF, produced by the gene immediately downstream of rifE, is thought to catalyse the intramolecular cyclization of the PKS product, thereby forming the ansamacrolide precursor of Rf B.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Resorcylic acid lactones from the ginseng pathogen Ilyonectria mors-panacis

Ilyonectria mors-panacis, previously Cylindrocarpon destructans, is the main plant pathogen responsible for the fungal disease ginseng root rot. This economically important disease, also called disappearing root rot, reduces crop yields by an average of 30% at harvest. While the disease is well studied from ecological and genomic perspectives, the role of I. mors-panacis secondary metabolites in the disease process is not well understood. Our previous metabolomics study showed Ilyonectria strains that cause ginseng root rot produce mixtures of putative resorcylic acid lactones, whereas avirulent strains did not, and collectively synthesize fewer metabolites. To confirm these metabolomics findings, we isolated and characterized the secondary metabolites from I. mors-panacis DAOMC 251601, a strain that causes ginseng root rot. From its EtOAc soluble culture filtrate extract, eight resorcylic acid lactones (1-8), including chlorinated and non-chlorinated congeners, were characterized by HRMS and spectroscopic approaches (NMR, OR, UV). The structure of one new metabolite, named radicicol E (1), was elucidated and additional spectroscopic data for the known compound nordinonediol (2) are reported. Further, radicicol (9) production was confirmed by comparison to a standard. The roles that resorcylic acid lactones and the siderophore N,N′,N” triacetylfusarine C have in promoting Ilyonectria ginseng root rot are also discussed.     

Phytochemical and chemotaxonomic study on Berchemiella wilsonii (Schneid.) Nakai

Phytochemical investigation of the aerial parts of Berchemiella wilsonii (Schneid.) Nakai (Rhamnaceae) led to the isolation of four flavonoids (1–4), three phenolic acids (5–7), two megastigmane derivatives (8–9) and one triterpene (10). The structures of these compounds were elucidated as taxifolin (1), (−)-epicatechin (2), quercetin 3-O-a-l-arabinopyranoside (3), vitexin (4), methyl p-hydroxycinnamat (5), 3,4-dihydroxybenzoic acid (6), 2-hydroxy-4-methoxy-3,6-dimethyl benzoic acid (7), (3S,5R,6R,7E,9S)-3,5,6,9-tetrahydroxy-7-en-megastigmane (8), (6S,9R)-roseoside (9) and lupeol (10) on the basis of NMR spectral data and comparison with literature values. These results are the first chemical constituent data of the genus Berchemiella, and the chemotaxonomic significance of these compounds is discussed.     

Chemical constituents from the roots of Caragana grandiflora (M. B.) DC. and their chemotaxonomic importance

A comprehensive phytochemical research on roots of Caragana grandiflora, a native plant to Iran, resulted in isolation of ten compounds including four phenolic compounds (2, 4, 5, 8), two fatty alcohols (1, 6), one fatty acid (9), one triterpene (3), one glyceride derivatives (7) and one fatty acid methyl ester (10), from which eight compounds (1, 2, 4–6 and 8–10) were isolated from the genus Caragana and two compounds (5 and 10) from the family Fabaceae, for the first time. All compounds (1–10) were described from Caragana grandiflora for the first time. Chemical structures of the purified compounds were identified through FT-IR, NMR and MSS, and spectral data comparison with literature reported evidences.Our findings provide valuable information in reporting the rare existence of natural fatty acid methyl ester (10) in the Fabaceae family. Moreover, the chemotaxonomic significance of these compounds was discussed.     

Two new quinic acid derivatives from the fruits of Chaenomeles speciosa

Two new quinic acid derivatives (1, 2), together containing eighteen (3–20) known compounds, were isolated from the fruits of Chaenomeles speciosa. Spectroscopic methods and previous data retrieved from the literature were used to determine the chemical structures of the compounds. Among the compounds, quinic acid derivatives (3, 4, 6, 7), phenolic acid compounds (8, 10, 11) and catechin derivatives (18, 19, 20) were isolated for the first time from the family Chaenomeles. The chemotaxonomic significance of the compounds was also discussed.     

Pentaketide resorcylic acid synthesis by type III polyketide synthase from Neurospora crassa

Type III polyketide synthases (PKSs) are responsible for aromatic polyketide synthesis in plants and bacteria. Genome analysis of filamentous fungi has predicted the presence of fungal type III PKSs, although none have thus far been functionally characterized. In the genome of Neurospora crassa, a single open reading frame, NCU04801.1, annotated as a type III PKS was found. In this report, we demonstrate that NCU04801.1 is a novel type III PKS catalyzing the synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named 2'-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a starter substrate and condensed four molecules of malonyl-CoA to give a pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic acids, aldol condensation and aromatization of the intermediate, which is still attached to the enzyme, are presumably followed by hydrolysis for release of the product as a resorcylic acid. ORAS is the first type III PKS that synthesizes pentaketide resorcylic acids.     

Discovery of N-(2,3,5-triazoyl)mycophenolic amide and mycophenolic epoxyketone as novel inhibitors of human IMPDH

Syntheses of ten derivatives of mycophenolic acid (MPA) at C-6′ position, and structure–activity relationship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of N-(2,3,5-triazolyl)mycophenolic amide 4, (7′S) mycophenolic epoxyketone 9 and (7′R) mycophenolic epoxyketone 10 having potent inhibitory activity against human inosine-5′-monophosphate dehydrogenase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds 4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and 10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 μM. These observations suggest that the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of HDAC.     

Phenolic constituents from the roots of Rosa laevigata (Rosaceae)

Chemical investigation of the root of Rosa laevigata led to the isolation of sixteen phenolic compounds, including seven flavonoids (1–7), five condensed tannins (8–12), two stilbenes (13 and 14) and two benzoic acid derivatives (15 and 16). Their structures were identified as (+)-catechin (1), (+)-gallocatechin (2), (2R, 3S, 4S)-cis- leucocyanidin (3), (2R, 3S, 4S)-cis-leucofisetinidin (4), (2S, 3R, 4R)-cis- leucofisetinidin (5), dehydrodicatechin A (6), phloridzin (7), procyanidin B3 (8), fisetinidol-(4α, 8)-catechin (9), guibourtinidol- (4α, 8)-catechin (10), ent- isetinidol -(4α, 6)-catechin (11), fisetinidol-(4β, 8)-catechin (12), (Z)-3-methoxy-5-hydroxy- stilbene (13), (Z)-piceid (14), gallic acid (15) and 4-hydroxybenzoic acid- 4-O-β-D-glucopyranoside (16). Among them, compounds 3–7, 9–14, and 16 were isolated from R. laevigata for the first time, and compounds 3–7, 9, 10, 12–14 and 16 were reported for the first time from the genus Rosa. The chemotaxonomic significance of these compounds was summarized.     

Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana

Natural inhibitors of fatty acid synthase (FAS) are emerging as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Garcinia mangostana led to the isolation of 13 phenolic compounds (1–13) mainly including xanthone and benzophenone, in which compounds 7, 8, 9, 10, and 11 were isolated from this plant for the first time and compound 9 was a new natural product. These isolates possess strong inhibitory activity of FAS with the IC₅₀ values ranging from 1.24 to 91.07 μM. The study indicates that two types of natural products, xanthones and benzophenones, could be considered as promising FAS inhibitors.     

Phytochemical and chemotaxonomic study on Pholidota pallida lindl

Phytochemical investigation of Pholidota pallida Lindl. led to the isolation of eleven compounds 1–11 (coelonin 1, lusianthridin 2, flavanthrin 3, batatasin-Ⅲ 4, 3′,5-dihydroxy-2-(4-hydroxybenzyl)-3-methoxybibenzyl 5, gigantol 6, 3-[2-(3-hydroxyphenyl) ethyl]-2,4-bis[(4-hydroxyphenyl) methyl]-5-methoxyphenol 7, hydroxytyrosyl butyrate 8, (24R)-ethylcholest-5-en-3-ol-7-one 9, taraxerone 10, friedelin 11) including three phenanthrenes 1–3, four bibenzyls 4–7, one hydroxytyrosyl 8, one steroid 9 and two terpenoids 10–11. The structures of these compounds were elucidated by spectroscopic analyses. This is the first report of isolation of compounds 1–11 from Pholidota pallida and compounds 5 and 8–11 within genus Pholidota. Compound 8 is a new natural product, isolated from a natural source for the first time. Furthermore, the chemotaxonomic significance of the isolates was also discussed.     

Chemical constituents of Medinilla septentrionalis (W. W. Sm.) H. L. Li (Melastomataceae)

Our phytochemical investigation of the whole plants of Medinilla septentrionalis (W. W. Sm.) H. L. Li led to the isolation of five tannins (1–5), five phenolic acids and phenolic acid derivatives (6–10), four flavonoids (11–14), two triterpenes (15 and 16), and one hydantoin derivative (17). The structures of the obtained compounds were identified using spectrometric methods (¹H NMR, ¹³C NMR and MS). This is the first study reporting on the chemical constituent of M. septentrionalis and the chemotaxonomic relationships between Medinilla and other genera of Melastomataceae.     

Biotransformation of ent-pimaradienoic acid by cell cultures of Aspergillus niger

Microbial transformation stands out among the many possible semi-synthetic strategies employed to increase the variety of chemical structures that can be applied in the search for novel bioactive compounds. In this paper we obtained ent-pimaradienoic acid (1, PA, ent-pimara-8(14),15-dien-19-oic acid) derivatives by fungal biotransformation using Aspergillus niger strains. To assess the ability of such compounds to inhibit vascular smooth muscle contraction, we also investigated their spasmolytic effect, along with another five PA derivatives previously obtained in our laboratory, on aortic rings isolated from male Wistar rats. The microbial transformation experiments were conducted at 30 °C using submerged shaken liquid culture (120 rpm) for 10 days. One known compound, 7α-hydroxy ent-pimara-8(14),15-dien-19-oic acid (2), and three new derivatives, 1β-hydroxy ent-pimara-6,8(14),15-trien-19-oic acid (3), 1α,6β,14β-trihydroxy ent-pimara-7,15-dien-19-oic acid (4), and 1α,6β,7α,11α-tetrahydroxy ent-pimara-8(14),15-dien-19-oic acid (5), were isolated and identified on the basis of spectroscopic analyses and computational studies. The compounds obtained through biotransformation (2–5) did not display a significant antispasmodic activity (values ranging from 0% to 16.8% of inhibition); however the previously obtained diterpene, methyl 7α-hydroxy ent-pimara-8(14),15-dien-19-oate (8), showed to be very effective (82.5% of inhibition). In addition, our biological results highlight the importance to study the antispasmodic potential of a large number of novel diterpenes, to conduct further structure–activity relationship investigations.     

Chemical constituents of Bulbophyllum wendlandianum (Kraenzl.) Dammer and their chemotaxonomic significance

Phytochemical investigation of Bulbophyllum wendlandianum (Kraenzl.) Dammer led to the isolation of twenty-three compounds 1–23 (flavanthrinin 1, coelonin 2, lusianthridin 3, densiflorol B 4, plicatol B 5, batatasin-lll 6, gigantol 7, 5-hydroxy-3,3′-dimethoxy-2-(p-hydroxybenzyl) bibenzyl 8, 2,2-dimethyl-5-hydroxy-6-carboxy-7-(2-phenylethyl) 9, tristin 10, p-hydroxybenzyl ethyl ether 11, p-hydroxybenzaldehyde 12, hydroquinone 13, coniferaldehyde 14, p-hydroxybenzyl alcohol 15, 3,4-dihydroxy benzaldehyde 16, stigmasterol 17, β-sitosterol 18, ergosterol peroxide 19, (+)pinoresinol 20, n-butyl sulfoxide 21, tridec-4E-en-l-yl acetate 22, ethyl linolate 23) including five phenanthrenes 1–5, five bibenzyls 6–10, six phenols 11–16, three sterols 17–19, one lignan 20, one n-butyl sulfoxide 21 and two fatty acids 22–23. The structures of these compounds were elucidated by spectroscopic analyses. This is the first report of isolation of compounds 1–23 from Bulbophyllum wendlandianum and compounds 8–9, 11, 13, 15–16 and 19–23 within genus Bulbophyllum. Compound 21 is a new natural product, isolated from a natural source for the first time. Furthermore, the chemotaxonomic significance of the isolates was also discussed.     

Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.     

Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.     

Synthesis and anti-tumor evaluation of novel 25-hydroxyprotopanaxadiol analogs incorporating natural amino acids

In the current study, derivatives of 25-hydroxyprotopanaxadiol (25-OH-PPD) were prepared and their in vitro anti-tumor activities were tested on six different human tumor cell lines by standard MTT assay. The results showed that combining an ester group combined with the presence of an amino acid moiety led to a 10-fold improved anti-tumor activity. Compound 1c exhibited the best anti-tumor activity in the in vitro assays. Compounds 2c, 3c, 4c, 5c, 6c and 8b showed better anti-tumor activities compared to the parent compound 25-OH-PPD. The current results may provide useful data for researching and developing new anti-cancer agents.     

Chemical constituents of the roots of Scorzonera divaricata and their chemotaxonomic significance

The phytochemical study of the roots of Scorzonera divaricata Turcz led to the isolation of 27 compounds, including eight sterols (1–8), one lignan (9), two cumarins (10, 11), five phenylpropanoids (12–16), six benzene derivatives (17–22), methyl-β-D-fructofuranoside (23), monolinolein (24), and three aliphatic acids (25–27). The structures of isolated compounds were identified using NMR and ESI-MS spectroscopic methods and comparing them with those previously reported. Except for β-daucosterol (8), scopoletin (10) and caffeic acid (16) from S. divaricata, this is the first report of the other 24 compounds from S. divaricata. Among them, eleven compounds (2–6, 11, 17, 19, 20, 23, 25) were reported from genus Scorzonera for first time, suggesting that they could be used to distinguish S. divaricata from the other species of Scorzonera. Furthermore, the chemotaxonomic significance of isolated compounds from S. divaricata has also been discussed.     

Chemical variation from the neoantimycin depsipeptide assembly line

Here we report the biosynthetic pathway for the neoantimycin and present three novel neoantimycin analogues, neoantimycin D (1), E (2) and F (3), from this assembly system from Streptoverticillium orinoci. Identification of these novel neoantimycin variants was achieved by selective MS/MS interrogation of natural product extracts using diagnostic fragments of the known neoantimycins. Their structures, including the absolute configurations, were elucidated using a combination of NMR experiments, detailed MS/MS experiments and the advanced Marfey’s method. The biosynthetic pathway of neoantimycin was dissected by genome sequencing data analysis for the first time, which includes a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly lines.