Nonactin biosynthesis: Setting limits on what can be achieved with precursor-directed biosynthesis

Nonactin, produced by Streptomyces griseus ETH A7796, is a macrotetrolide assembled from nonactic acid. It is an effective inhibitor of drug efflux in multidrug resistant erythroleukemia K562 cells at sub-toxic concentrations and has been shown to possess both antibacterial and antitumor activity. As total synthesis is impractical for the generation of nonactin analogs we have studied precursor-directed biosynthesis as an alternative as it is known that nonactic acid can serve as a nonactin precursor in vivo. To determine the scope of the approach we prepared and evaluated a furan-based nonactic acid derivative, 11. Although no new nonactin analogs were detected when 11 was administered to S. griseus fermentative cultures, a significant inhibition of nonactin biosynthesis was noted (IC₅₀ ～ 100 μM). Cell mass, nonactic acid production and the generation of other secondary metabolites in the culture were unaffected by 11 demonstrating that 11 selectively inhibited the assembly of nonactin from nonactic acid. While we were unable to generate new nonactin analogs we have discovered, however, a useful inhibitor that we can use to probe the mechanism of nonactin assembly with the ultimate goal of developing more successful precursor-directed biosynthesis transformations.     

Determination of ionophore antibiotics nactins produced by fecal Streptomyces from sheep

To investigate the correlation between fecal actinobacteria and host animals, Streptomyces was isolated from fresh faeces of healthy sheep and secondary metabolites were analyzed. The most frequently isolated strain S161 with antibiotic activity against bacteria and fungi were analyzed. The S161 showed the highest 99 % similarity to Streptomyces canus DSB17 based on the 16S rRNA gene sequence analysis. Metabolite analysis based on MS and NMR spectra showed that S161 produces nactins, cyclotetralactones derived from nonactic acid and homononactic acid as building units of ionophoretic character. Due to ionophores are antimicrobial compounds that are commonly fed to ruminant animals to improve feed efficiency, stable beneficial interactions between Streptomyces bacteria and vertebrates have been demonstrated.     

New Derivatives of Nonactic and Homononactic Acids from Bacillus pumilus Derived from Breynia fruticosa

Six new nonactic and homononactic acid derivatives, ethyl homononactate ( 1 ), ethyl nonactate ( 2 ), homononactyl homononactate ( 6 ), ethyl homononactyl nonactate ( 7 ), ethyl homononactyl homononactate ( 8 ), and ethyl nonactyl nonactate ( 9 ), as well as four known compounds, homononactic acid ( 3 ), nonactic acid ( 4 ), homononactyl nonactate ( 5 ), and bishomononactic acid ( 10 ), were isolated from culture broth of Bacillus pumilus derived from Breynia fruticosa. The structures of new compounds were elucidated by spectroscopic analysis and chemical methods. The optical purities of 1 – 6 were determined by HPLC/MS after treatment with L ‐phenylalanine methyl ester. The dimeric compounds 5 – 9 showed weak cytotoxic activities against five human cancer cell lines (IC₅₀ 19–100 μg/ml).     

Microbial Degradation of a Macrotetrolide Miticide in Soil

Macrotetrolide, a miticide consisting of tetranactin, trinactin, and dinactin, was readily biodegradable and hence did not accumulate in soil. [U-¹⁴C]macrotetrolide was rapidly degraded via its constituent hydroxycarboxylic acids to carbon dioxide and water. In culture media, however, the mixture was hydrolyzed to homononactic and nonactic acids by three strains of Bacillus sp. and two of Micrococcus sp. The latter strains were able to hydrolyze 500 μg of the antibiotic per ml within a few days and to grow in the presence of 4,000 μg of the antibiotic per ml. However, they were unable to assimilate the constituent acids which accumulated in the culture medium.     

Production of macrolide antibiotics from a cytotoxic soil <Emphasis Type="Italic">Streptomyces</Emphasis> sp. strain ZDB

Crude extract from a culture of a soil Streptomyces sp. strain ZDB showed toxicity towards Artemia salina and antimicrobial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Chlorella vulgaris, and Chlorella sorokiniana. Large scale fermentation of the strain led to the isolation of the macrolide antibiotics, bafilomycins A1 (1), B1 (2), and D (3) together with nonactic acid (4) and bostrycoidin-9-methyl ether (5). Structures of the antibiotics were determined based on spectral data analysis. We describe the isolation of the compounds and characterization of the producing strain.     

A 'bacteriocin PCR array' for identification of bacteriocin-related structural genes in lactic acid bacteria

Bacteriocins have been identified in many strains of lactic acid bacteria (LAB) which are a source of natural food preservatives and microbial inhibitors. Our objectives were to use a PCR array of primers to identify bacteriocin structural genes in Bac⁺ LAB. DNA sequence homology at the 5′- and 3′-ends of the various structural genes indicated that non-specific priming may allow PCR amplification of heterologous bacteriocin genes. Successful amplification was obtained by real-time PCR and confirmed by melting curve and agarose gel analysis. Sequence information specific to targeted bacteriocin structural genes from the intra-primer regions of amplimers was compared to sequences residing in GenBank. The bacteriocin PCR array allowed the successful amplification of bacteriocin structural genes from strains of Lactobacillus, Lactococcus, and Pediococcus including one whose amino acid sequence was unable to be determined by Edman degradation analysis. DNA sequence analysis identified as many as 3 bacteriocin structural genes within a given strain, identifying ten unique bacteriocin sequences that were previously uncharacterized (partial homology) and one that was 100% identical to sequences in GenBank. This study provides a rapid approach to sequence and identify bacteriocin structural genes among Bac⁺ LAB using a microplate bacteriocin PCR array.     

Amino acids and low-molecular-weight carbohydrates of some marine red algae

Amino acids and low-MW carbohydrates of 18 red algae have been analyzed. Several non-protein amino acids have been identified, including pyrrolidine-2,5-dicarboxylic acid (3c) and N-methylmethionine sulfoxide (5), new natural products, and 13 known compounds, citrulline, β-alanine, γ-aminobutyric acid, baikiain (1), pipecolic acid (2), domoic acid (3a), kainic acid (3b), azetidine-2-carboxylic acid (4), methionine sulfoxide taurine, N-methyltaurine, N,N-dimethyltaurine and N,N,N-trimethyltaurine. Sugars present were mainly floridoside, isofloridoside and mannoglyceric acid. Details of the structural elucidation of new compounds are also given.     

Synthesis of aminoacylated N6,N6-dimethyladenosine solid support for efficient access to hydrolysis-resistant 3′-charged tRNA mimics

RNA-amino acid and RNA-peptide conjugates that mimic charged tRNA 3′-ends are valuable substrates for structural and functional investigations of ribosomal complexes. To obtain such conjugates, most synthetic approaches that are found in the literature make use of puromycin. This well available aminonucleoside antibiotic contains a dimethylamino group at the nucleobase and a methylated tyrosine that is connected via an amide linkage to the ribose moiety. To increase structural diversity, we present the synthesis of a N⁶,N⁶-dimethylated 3′-azido-3′-deoxyadenosine precursor that can be coupled to any amino acid. Further derivatization results in the solid support that is eligible for the preparation of stable 3′-aminoacyl- or 3′-peptidyl-tRNA termini with an amide instead of the natural ester linkage. The present work expands our previously established route that delivered a broad range of peptidyl-tRNA mimics to the corresponding counterparts with N⁶,N⁶-dimethylation pattern of the terminal adenosine (A76). This aspect is of significance to modulate the binding preferences of the mimics for ribosomal A- versus P-site.     

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin

The myotoxic mechanism for PLA₂-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA₂-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA₂-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA₂-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA₂-like toxins in general.     

Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe³⁺. Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(−)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.     

A Combination of Structural and Cis-Regulatory Factors Drives Biochemical Differences in Drosophila melanogaster Malic Enzyme

The evolutionary significance of molecular variation is still contentious, with much current interest focusing on the relative contribution of structural changes in proteins versus regulatory variation in gene expression. We present a population genetic and biochemical study of molecular variation at the malic enzyme locus (Men) in Drosophila melanogaster. Two amino acid polymorphisms appear to affect substrate-binding kinetics, while only one appears to affect thermal stability. Interestingly, we find that enzyme activity differences previously assigned to one of the polymorphisms may, instead, be a function of linked regulatory differences. These results suggest that both regulatory and structural changes contribute to differences in protein function. Our examination of the Men coding sequences reveals no evidence for selection acting on the polymorphisms, but earlier work on this enzyme indicates that the biochemical variation observed has physiological repercussions and therefore could potentially be under natural selection.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.     

Efficacy of artificial humic acid as a selective nutrient in HV agar used for the isolation of soil actinomycetes

The efficacies of various kinds of humic acid, as the source of carbon and nitrogen in HV agar reported in the previous paper, were compared to the selective isolation of soil actinomycetes. The 4 types of natural humic acid, A, B, P and Rp were prepared from different soils, and 3 kinds of artificial humic acid were made from carbohydrates and urea in our laboratory.Among the natural humic acids, type Rp, which has been reported to be associated with an initial state of humification in natural conditions, showed the greatest efficacy.However, one of the artificial humic acids, which was prepared from glucose and urea, was considered to be superior to the Rp natural humic acid: 1) The HV agar containing this artificial humic acid (HV-glucose HA agar) produced the same large number of actinomycete colonies on the plate as that of the HV agar with type Rp soil humic acid (HV-Rp agar). 2) The HV-glucose HA agar restricted the number of bacterial colonies on the plates to one-half of that on HV-Rp agar plates. 3) The quality of natural himic acids varies, whereas artificial ones are more constant and can be made in any laboratory.     

Non-food bioactive products for insecticides (II): Investigation on stress responses of Tetranychus cinnabarinus Boisduval against a derivative of the alkaloid matrine

Besides structural modification of natural bioactive products to afford promising agrochemical candidates, investigation of their mechanisms of action against pests is also an important strategy to obtain novel potentially botanical pesticides. N-(p-Ethyl)phenylsulfonylmatrinic acid (2), derived from an natural alkaloid matrine (1), exhibited about 5.9-fold more pronounced acaricidal activity than 1 against the adult females of Tetranychus cinnabarinus Boisduval, and good control efficiency in the greenhouse. By comparison of nAChR, AChE and VGSC of treated and untreated T. cinnabarinus via RT-PCR and qRT-PCR analysis, it was found that compound 2 could activate nAChR and VGSC via up-regulation of nAChR α1, α4 and α5 subunits and VGSC expressions; compound 2 may be the AChE and AChE enzyme inhibitor. Importantly, a scheme of compound 2 interaction with nAChR, AChE and VGSC of T. cinnabarinus was proposed. It will lay the foundation for future optimization and application of matrine derivatives as agrochemicals.     

A structural analysis of the natural egress of Toxoplasma gondii

Previous studies have analysed the process of Toxoplasma gondii egress with the aid of inducers, such as calcium ionophores. Although calcium transients have been successful in triggering T. gondii egress, the structural panorama of “natural” and artificial events should match. The present study approaches the natural egress of this parasite using super-resolution and electron microscopy and reveals lytic and non-lytic events of individual egress; this corroborates the use of calcium ionophore as a reliable tool to trigger parasite egress. Altogether, our data suggest that different signalling routes can converge to similar structural aspects in natural and induced egress.     

Spectroscopic characterization of (57)Fe-enriched cytochrome c

Investigation of the heme iron dynamics in cytochrome c with Mössbauer spectroscopy and especially nuclear resonance vibrational spectroscopy requires the replacement of the natural abundant heme iron with the ⁵⁷Fe isotope. For demetallization, we use a safer and milder ferrous sulfate–hydrochloric acid method in addition to the harsher commonly used hydrofluoric acid-based procedure. The structural integrity of the ⁵⁷Fe-reconstituted protein in both oxidation states is confirmed from absorption spectra and a detailed analysis of the rich resonance Raman spectra. These results reinforce the application of metal-substituted heme c proteins as reliable models for the native proteins.     

A natural propenoic acid derivative activates peroxisome proliferator-activated receptor-β/δ (PPARβ/δ)

AimsPrevious studies showed that natural prenyloxyphenylpropanoid derivatives have potent biological properties in vivo. Given the structural similarities between these compounds and known peroxisome proliferator-activated receptor (PPAR) agonists, the present study examined the hypothesis that propenoic acid derivatives activate PPARs.Main methodsChimeric reporter assays were performed to identify propenoic acid derivates that could activate PPARs. Quantitative polymerase chain reaction (qPCR) analysis of wild-type and Pparβ/δ-null mouse primary keratinocytes was performed to determine if a test compound could specifically activate PPARβ/δ. A human epithelial carcinoma cell line and primary mouse keratinocytes were used to determine the effect of the compound on cell proliferation.Key findingsThree of the propenoic acid derivatives activated PPARs, with the greatest efficacy being observed with prenyloxycinnamic acid derivatives 4′-geranyloxyferulic acid (compound 1) for PPARβ/δ. Compound 1 increased expression of a known PPARβ/δ target gene through a mechanism that requires PPARβ/δ. Inhibition of cell proliferation by compound 1 was found in a human epithelial carcinoma cell line.SignificanceResults from these studies demonstrate that compound 1 can activate PPARβ/δ and inhibit cell proliferation of a human skin cancer cell line, suggesting that the biological effects of 4′-geranyloxyferulic acid may be mediated in part by activating this PPAR isoform.