RESUMO
Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.
Assuntos
Benzodiazepinas/metabolismo , Lincomicina/biossíntese , Oxirredutases/metabolismo , Pirróis/metabolismo , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Catálise , Depsipeptídeos/biossíntese , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Lincomicina/química , Lincomicina/farmacologia , Modelos Moleculares , Oxirredutases/química , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Prolina/análogos & derivados , Prolina/metabolismo , Pirróis/química , Pirróis/farmacologia , Riboflavina/análogos & derivados , Riboflavina/química , Riboflavina/metabolismo , Especificidade por Substrato , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The effects in vitro of 2,3-dehydrosilybin and several galloyl esters and methyl ethers on the viability, proliferation, and migration of human umbilical vein endothelial cells (HUVECs) were evaluated. The monogalloyl esters were synthesized by a chemoselective esterification method or by Steglich esterification of suitably protected 2,3-dehydrosilybin (1) with protected gallic acid. 2,3-Dehydrosilybin (1) displayed more potent cytotoxic, antiproliferative, and antimigratory activities (IC50 12.0, 5.4, and 12.2 µM, respectively) than silybin. The methylated derivatives were less active, with the least potent being 3,7-di-O-methyl-2,3-dehydrosilybin (6). On the other hand, galloylation at C-7 OH and C-23 OH markedly increased the cytotoxicity and the effects on the proliferation and migration of HUVECs. The most active derivative was 7-O-galloyl-2,3-dehydrosilybin (13; IC50 value of 3.4, 1.6, and 4.7 µM in the cytotoxicity, inhibition of proliferation, and antimigratory assays, respectively). Overall, this preliminary structure-activity relationship study demonstrated the importance of a 2,3-double bond, a C-7 OH group, and a galloyl moiety in enhancing the activity of flavonolignans toward HUVECs.
Assuntos
Silimarina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sequestradores de Radicais Livres/química , Ácido Gálico/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Éteres Metílicos/farmacologia , Estrutura Molecular , Silibina , Silimarina/química , Relação Estrutura-AtividadeRESUMO
A new, efficient, and general semisynthesis of hydnocarpin-type flavonolignans was developed and optimized, enabling gram-scale production of hydnocarpin D (2). Moreover, the syntheses of optically pure hydnocarpin isomers [(10R,11R)-hydnocarpin (1a), (10R,11R)-hydnocarpin D (2a), and (10S,11S)-hydnocarpin D (2b)], as well as the synthesis of isohydnocarpin (8), were achieved for the first time utilizing this new method. The synthesis is based on the two-step transformation of the readily available flavonolignans from milk thistle (Silybum marianum), accessible by isolation from the commercial extract silymarin. The first step relies on the regioselective formylation of the C-3 hydroxy group of the dihydroflavonol-type precursor using the Vilsmeier-Haack reagent, followed by formic acid elimination by triethylamine in the second step. The synthesized compounds were effective inhibitors of Staphylococcus aureus biofilm formation, with (10S,11S)-hydnocarpin D (2b) being the most potent inhibitor. Furthermore, the effect of glucose on biofilm formation was tested, and glucose decreased the biofilm inhibitory activity of 2b. Moreover, 2b increased the susceptibility of Staph. aureus to enrofloxacin.
Assuntos
Biofilmes/efeitos dos fármacos , Flavonolignanos/isolamento & purificação , Flavonolignanos/farmacologia , Silybum marianum/química , Staphylococcus aureus/efeitos dos fármacos , Antioxidantes , Cromatografia Líquida de Alta Pressão , Flavonolignanos/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Silimarina/química , Relação Estrutura-AtividadeRESUMO
Methods were developed and optimized for the preparation of the 2,3-cis- and the 10,11-cis-isomers of silybin by the Lewis acid catalyzed (BF3âOEt2) isomerization of silybins A (1a) and B (1b) (trans-isomers). The absolute configuration of all optically pure compounds was determined by using NMR and comparing their electronic circular dichroism data with model compounds of known absolute configurations. Mechanisms for cis-trans-isomerization of silybin are proposed and supported by quantum mechanical calculations.
RESUMO
Natural polyphenols are known to be oxidized by free radicals, which partially explains the antioxidant properties of a number of these compounds. This oxidation may also be used to synthesise new compounds of biological interest, for example, dimers. The present theoretical study describes the existing experimental evidence showing that silybin and dehydrosilybin [natural polyphenols isolated from milk thistle (Silybum marianum)] form dimers regioselectively. Based on DFT calculations, thermodynamic and kinetic values were computed in order to better understand the physicochemical behaviour of these dimerisation processes. Calculations showed that after H-atom transfer (from polyphenol to radical), dimerisation could proceed in two steps: 1) bond formation and, when possible, 2) tautomerisation reorganisation. The former step is the limiting step, while the latter is crucial for the process to be thermodynamically favourable (ΔG<0). If this rearrangement is impossible then dimerisation is not feasible, or at least becomes a minor process (e.g., dehydrosilybin dimerisation after H-atom abstraction from the 3-OH group). This explains the regioselectivity of polyphenol dimerisation.
Assuntos
Flavonolignanos/química , Silimarina/química , Dimerização , Cinética , Oxirredução , Polifenóis/química , Silibina , Estereoisomerismo , TermodinâmicaRESUMO
Two chromatographic narrow-bore columns, a novel 2.6 µm particle-packed Kinetex™ C18 core-shell (50×2.1 mm id) and monolithic Chromolith(®) FastGradient RP-18e (50×2 mm id), were evaluated for the analysis of diastereoisomers of the flavonolignans silybin and 23-O-acetylsilybin under isocratic conditions. The main advantages of the core-shell column are markedly higher efficiency (hmin =2.8 versus 5.6 for silybin A) and better peak symmetry. The Kinetex column exhibits only a slight change in the height equivalent of the theoretical plate with a higher linear velocity of the mobile phase. The monolithic column shows notably higher selectivity in terms of selectivity factor (1.21 versus 1.12) in the analysis of critical-pair of diastereoisomers (silybin A and silybin B) and enables shorter run duration (approx. twofold) together with lower backpressure. The resolution power was found to be comparable, but the Kinetex column required a higher pressure of the mobile phase that, together with the higher chance of clogging, can be a disadvantage in the separation of biological samples. Successful baseline separation of silybin diastereoisomers in real pharmaceutical sample on monolithic column was accomplished.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Resinas Sintéticas/química , Silimarina/química , Cromatografia Líquida de Alta Pressão/instrumentação , Silibina , Silimarina/isolamento & purificação , EstereoisomerismoRESUMO
beta-N-Acetylhexosaminidases feature so-called wobbling specificity, which means that they cleave substrates both in gluco- and galacto- configurations, with the activity ratio depending on the enzyme source. Here we present the new finding that fungal beta-N-acetylhexosaminidases are able to hydrolyze and transfer 4-deoxy-N-acetylhexosaminides with high yields. This clearly demonstrates that the 4-hydroxy moiety at the substrate pyranose ring is not essential for substrate binding to the enzyme active site, which was also confirmed by molecular docking of the tested compounds into the model of the active site of beta-N-acetylhexosaminidase from Aspergillus oryzae. A set of four 4-deoxy-N-acetylhexosaminides was synthesized and screened against a panel of beta-N-acetylhexosaminidases (extracellular and intracellular) from various sources (fungal, human, animal, plant and bacterial) for hydrolysis. The results of this screening are reported here, as well as the structures of three novel 4'-deoxy-disaccharides prepared by transglycosylation reaction with high yields (52% total disaccharide fraction) using beta-N-acetylhexosaminidase from Talaromyces flavus.
Assuntos
Desoxiglucose/análogos & derivados , Glucosamina/análogos & derivados , beta-N-Acetil-Hexosaminidases/metabolismo , Desoxiglucose/síntese química , Desoxiglucose/química , Glucosamina/síntese química , Glucosamina/química , Estrutura Molecular , Estereoisomerismo , Especificidade por Substrato , beta-N-Acetil-Hexosaminidases/químicaRESUMO
Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.
Assuntos
Glicoconjugados/síntese química , Glicoconjugados/metabolismo , Leucócitos Mononucleares/imunologia , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Glicoconjugados/química , Glicosilação , Humanos , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/metabolismo , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Oligopeptídeos/química , Oximas/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismoRESUMO
The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-L-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4'-butyl-4'-depropyllincomycin and 4'-pentyl-4'-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4'-pentyl-4'-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Lincomicina/metabolismo , Lincomicina/farmacologia , Staphylococcus/efeitos dos fármacos , Streptomyces/metabolismo , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Lincomicina/análogos & derivados , Lincomicina/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Prolina/análogos & derivados , Prolina/metabolismo , Infecções Estafilocócicas/microbiologia , Streptomyces/genéticaRESUMO
Reactive oxygen species (ROS) originating from mitochondria are perceived as a factor contributing to cell aging and means have been sought to attenuate ROS formation with the aim of extending the cell lifespan. Silybin and dehydrosilybin, two polyphenolic compounds, display a plethora of biological effects generally ascribed to their known antioxidant capacity. When investigating the cytoprotective effects of these two compounds in the primary cell cultures of neonatal rat cardiomyocytes, we noted the ability of dehydrosilybin to de-energize the cells by monitoring JC-1 fluorescence. Experiments evaluating oxygen consumption and membrane potential revealed that dehydrosilybin uncouples the respiration of isolated rat heart mitochondria albeit with a much lower potency than synthetic uncouplers. Furthermore, dehydrosilybin revealed a very high potency in suppressing ROS formation in isolated rat heart mitochondria with IC(50) = 0.15 µM. It is far more effective than its effect in a purely chemical system generating superoxide or in cells capable of oxidative burst, where the IC(50) for dehydrosilybin exceeds 50 µM. Dehydrosilybin also attenuated ROS formation caused by rotenone in the primary cultures of neonatal rat cardiomyocytes. We infer that the apparent uncoupler-like activity of dehydrosilybin is the basis of its ROS modulation effect in neonatal rat cardiomyocytes and leads us to propose a hypothesis on natural ischemia preconditioning by dietary polyphenols.
Assuntos
Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Silimarina/farmacologia , Análise de Variância , Animais , Benzimidazóis , Carbocianinas , Corantes Fluorescentes , Concentração Inibidora 50 , Estrutura Molecular , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Rotenona/toxicidade , Silimarina/químicaRESUMO
In nature, the flavonolignan silybin (1) occurs as a mixture of two diastereomers, silybin A and silybin B, which in a number of biological assays exhibit different activities. A library of hydrolases (lipases, esterases, and proteases) was tested for separating the silybin A and B diastereomers by selective transesterification or by stereoselective alcoholysis of 23-O-acetylsilybin (2). Novozym 435 proved to be the most suitable enzyme for the preparative production of both optically pure silybins A and B by enzymatic discrimination. Gram amounts of the optically pure substances can be produced within one week, and the new method is robust and readily scalable to tens of grams.
Assuntos
Hidrolases/metabolismo , Lipase/metabolismo , Silimarina/química , Bactérias/enzimologia , Técnicas de Química Combinatória , Enzimas Imobilizadas , Proteínas Fúngicas , Cinética , Silybum marianum/química , Estrutura Molecular , Silibina , Silimarina/síntese química , Silimarina/isolamento & purificação , EstereoisomerismoRESUMO
Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.
Assuntos
Antibacterianos/metabolismo , Enterococcus faecium/metabolismo , Lipoglicopeptídeos/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/metabolismo , Teicoplanina/análogos & derivados , Teicoplanina/metabolismo , Enterococos Resistentes à Vancomicina/metabolismo , Vancomicina/metabolismo , Parede Celular/microbiologia , Fluorescência , Glicopeptídeos/metabolismo , Lipoglicopeptídeos/química , Testes de Sensibilidade Microbiana , Ligação Proteica/fisiologia , Rodaminas/química , Coloração e Rotulagem , Teicoplanina/química , Vancomicina/químicaRESUMO
Flavonolignans from silymarin, the standardized plant extract obtained from thistle, exhibit various antioxidant activities, which correlate with the other biological and therapeutic properties of that extract. To highlight the mode of action of flavonolignans as free radical scavengers and antioxidants, 10 flavonolignans, selectively methylated at different positions, were tested in vitro for their capacity to scavenge radicals (DPPH and superoxide) and to inhibit the lipid peroxidation induced on microsome membranes. The results are rationalized on the basis of (i) the oxidation potentials experimentally obtained by cyclic voltammetry and (ii) the theoretical redox properties obtained by quantum-chemical calculations (using a polarizable continuum model (PCM)-density functional theory (DFT) approach) of the ionization potentials and the O-H bond dissociation enthalpies (BDEs) of each OH group of the 10 compounds. We clearly establish the importance of the 3-OH and 20-OH groups as H donors, in the presence of the 2,3 double bond and the catechol moiety in the E-ring, respectively. For silybin derivatives (i.e., in the absence of the 2,3 double bond), secondary mechanisms (i.e., electron transfer (ET) mechanism and adduct formation with radicals) could become more important (or predominant) as the active sites for H atom transfer (HAT) mechanism are much less effective (high BDEs).
Assuntos
Antioxidantes/química , Flavonolignanos/química , Hidrogênio/química , Modelos Químicos , Íons/química , Modelos Moleculares , Estrutura Molecular , Silibina , Silimarina/química , TermodinâmicaRESUMO
This review critically surveys the literature published mainly within this millennium on the new and emerging applications of silybin (pure, chemically defined substance) and silymarin (flavonoid complex from Silybum marianum - milk thistle seeds). These compounds used so far mostly as hepatoprotectants were shown to have other interesting activities, e.g. anticancer and canceroprotective and also hypocholesterolemic activity. These effects were demonstrated in a large variety of illnesses of different organs, e.g. prostate, lungs, CNS, kidneys, pancreas and also in the skin protection. Besides the cytoprotective activity of silybin mediated by its antioxidative and radical-scavenging properties also new functions based on the specific receptor interaction were discovered. These were studied on the molecular level and modulation of various cell-signaling pathways with silybin was disclosed--e.g. NF-kappaB, inhibition of EGFR-MAPK/ERK1/2 signaling, activity upon Rb and E2F proteins, IGF-receptor signaling. Proapoptotic activity of silybin in pre- and/or cancerogenic cells and anti-angiogenic activity of silybin are other important findings that bring silymarin preparations closer to respective application in the cancer treatment. Discovery of the inhibition and modulation of drug transporters, P-glycoproteins, estrogenic receptors, nuclear receptors by silybin and some of its new derivatives contribute further to the better understanding of silybin activity on the molecular level. Silymarin application in veterinary medicine is reviewed as well. Recent works using optically pure silybin diastereomers clearly indicate extreme importance of the use of optically active silybin namely in the receptor studies. Significance of silymarin and its components in the medicine is clearly indicated by an exponential growth of publications on this topic--over 800 papers in the last 5 years.
Assuntos
Silimarina/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Inibidores da Angiogênese/uso terapêutico , Doenças dos Animais/tratamento farmacológico , Animais , Anti-Inflamatórios/uso terapêutico , Anticarcinógenos/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Cardiotônicos/uso terapêutico , Colagogos e Coleréticos/uso terapêutico , Feminino , Humanos , Neoplasias Pulmonares/prevenção & controle , Masculino , Neoplasias da Próstata/tratamento farmacológico , Receptores de Estrogênio/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , SilibinaRESUMO
Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.
Assuntos
Monofosfato de Adenosina/química , Aminoácidos/química , Evolução Química , Modelos Químicos , Mutagênese Sítio-Dirigida , Proteínas/química , Proteínas/genética , Especificidade por SubstratoRESUMO
Anticancer pyrrolobenzodiazepines (PBDs) are one of several groups of natural products that contain unusual 4-alkyl-l-proline derivatives (APDs) in their structure. APD moieties of PBDs are characterized by high structural diversity achieved through unknown biosynthetic machinery. Based on LC-MS analysis of culture broths, feeding experiments, and protein assays, we show that APDs are not incorporated into PBDs in their final form as was previously hypothesized. Instead, a uniform building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent postcondensation steps are initiated by the introduction of an additional double bond catalyzed by a FAD-dependent oxidoreductase, which we demonstrated with Orf7 from anthramycin biosynthesis. The resulting double bond arrangement presumably represents a prerequisite for further modifications of the APD moieties. Our study gives general insight into the diversification of APD moieties of natural PBDs and provides proof-of-principle for precursor directed and combinatorial biosynthesis of new PBD-based antitumor compounds.
Assuntos
Antineoplásicos/química , Benzodiazepinas/química , Pirróis/química , Antineoplásicos/metabolismo , Benzodiazepinas/classificação , Benzodiazepinonas/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Química Farmacêutica , Estrutura Molecular , Pirróis/classificaçãoRESUMO
The immediate post-condensation steps in lincomycin biosynthesis are reminiscent of the mycothiol-dependent detoxification system of actinomycetes. This machinery provides the last proven lincomycin intermediate, a mercapturic acid derivative, which formally represents the 'waste product' of the detoxification process. We identified and purified new lincomycin intermediates from the culture broth of deletion mutant strains of Streptomyces lincolnensis and tested these compounds as substrates for proteins putatively involved in lincomycin biosynthesis. The results, based on LC-MS, in-source collision-induced dissociation mass spectrometry and NMR analysis, revealed the final steps of lincomycin biosynthesis, i.e. conversion of the mercapturic acid derivative to lincomycin. Most importantly, we show that deacetylation of the N'-acetyl-S-cysteine residue of the mercapturic acid derivative is required to 'escape' the detoxification-like system and proceed towards completion of the biosynthetic pathway. Additionally, our results, supported by l-cysteine-13C3, 15N incorporation experiments, give evidence that a different type of reaction catalysed by the homologous pair of pyridoxal-5'-phosphate-dependent enzymes, LmbF and CcbF, forms the branch point in the biosynthesis of lincomycin and celesticetin, two related lincosamides.
RESUMO
Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.
RESUMO
Silymarin, a standardized mixture of flavonolignans, or its major constituents could be effective for prevention and treatment of hepatic damage or skin cancer. However, their potential side effects, such as modulation of endocrine functions via the disruption of estrogen receptor (ER) and/or aryl hydrocarbon receptor (AhR) activation, are largely unknown. In the present study, we investigated impact of silymarin, its constituents and a series of their synthetic derivatives on ER- and AhR-mediated activities using in vitro reporter gene assays. We found that none of the compounds under study affected the AhR-mediated activity in rat hepatoma cells. Contrary to that, several compounds behaved as either partial or full ER agonists. Silymarin elicited partial ER activation, with silybin B being probably responsible for a majority of the weak ER-mediated activity of silymarin; silybin A and other flavonolignans were found to be inactive and potent ER agonist taxifolin is only a minor constituent of silymarin. To our knowledge, this is probably the first time, when receptor-specific in vitro effects of separated diastereomers have been demonstrated. In contrast to silymarin constituents, the synthetic silybin derivatives, potentially useful as chemoprotective agents, did not modulate the ER-mediated activity, with exception of 23-O-pivaloylsilybin. Interestingly, 7-O-benzylsilybin potentiated ER-mediated activity of 17beta-estradiol despite possessing no estrogenic activity. In conclusion, our data suggest that estrogenicity of some silymarin constituents should be taken in account as their potential side effect when considered as chemopreventive compounds. These results also stress the need to study biological activities of purified or synthesized diastereomers of silybin derivatives.
Assuntos
Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Luciferases/biossíntese , Luciferases/genética , Estrutura Molecular , Ratos , Silibina , Silimarina/química , Silimarina/farmacologia , EstereoisomerismoRESUMO
In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.