Metabolic Investigation and Auxiliary Enzyme Modelization of the Pyrrocidine Pathway Allow Rationalization of Paracyclophane-Decahydrofluorene Formation.

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare -decalin, cytochalasan, and fused 6/15/5 macrocycles.

Combinatory Library of Microorganisms in the Selection of Reductive Activity Applied to a Ketone Mixture: Unexpected Highlighting of an Enantioselective Oxidative Activity.

Biocatalytic processes are increasingly used in organic synthesis for the preparation of targeted molecules or the generation of molecular diversity. The search for the biocatalyst is often the bottleneck in the development of the process. We described a combinatorial approach for the selection of active strains from a library of microorganisms.

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis.

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines.

Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease.

Molecular crosstalk between the endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum - Modulation of lipoxygenase activity and beauvericin production during the interaction.

Plants comprise many asymptomatic fungal endophytes with potential roles of plant protection against abiotic and biotic stresses. Endophytes communicate with their host plant, with other endophytes and with invading pathogens but their language remains largely unknown. This work aims at understanding the chemical communication and physiological interactions between the fungal endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum.

A Reactive Eremophilane and Its Antibacterial 2(1H)-Naphthalenone Rearrangement Product, Witnesses of a Microbial Chemical Warfare.

Two sesquiterpenes, 4-epi-microsphaeropsisin (1) and a dihydrofurano-2(1H)-naphthalenone (variabilone, 2) which represents a new skeleton, were isolated from endophytic fungus Paraconiothyrium variabile. Reactivity studies showed that eremophilane 1 is a precursor of 2 through acid-promoted methyl 1,2-migration and aromatization. An electrophilic intermediate of this transformation was intercepted by N-acetylcysteamine, a biomimetic nucleophile.

Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis.

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.

Talaroketals A and B, unusual bis(oxaphenalenone) spiro and fused ketals from the soil fungus Talaromyces stipitatus ATCC 10500.

Two novel oxaphenalenone dimers, talaroketals A () and B (), were isolated from the soil fungus Talaromyces stipitatus. Their structures and absolute configurations were determined on the basis of spectroscopic analyses, X-ray diffraction experiments and electronic circular dichroism. Compound () features a rare benzannulated 5,6-spiroketal ring system within the dimeric bis(oxaphenalenone) skeleton while the parent compound () harbors a fused bicyclic furano-pyran moiety.

Unexpected talaroenamine derivatives and an undescribed polyester from the fungus Talaromyces stipitatus ATCC10500.

Chemical investigation of the fungus Talaromyces stipitatus ATCC 10500, whose genome has been sequenced, led to the isolation of four undescribed talaroenamines B-E along with the known talaroenamine A. Their structures were elucidated on the basis of spectroscopic studies including mass spectrometry, extensive 2D NMR and electronic circular dichroism (ECD). Interestingly, talaroenamine A had previously been isolated from the strain of T.

Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a.

Synthesis, configuration assignment, and simultaneous quantification by liquid chromatography coupled to tandem mass spectrometry, of dihydroanatoxin-a and dihydrohomoanatoxin-a together with the parent toxins, in axenic cyanobacterial strains and in environmental samples.

We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra.

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid.

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed.

Insights into the reaction mechanism of the prolyl-acyl carrier protein oxidase involved in anatoxin-a and homoanatoxin-a biosynthesis.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins as well as the in-vitro reconstitution of the first steps of this biosynthesis. We now report experimental evidence supporting the proposed reaction mechanism of AnaB, a flavoprotein homologous to acyl-CoA dehydrogenase.

Unprecedented anthranilate priming involving two enzymes of the acyl adenylating superfamily in aurachin biosynthesis.

Biosynthesis of many polyketide-derived secondary metabolites is initiated by incorporating starter units other than acetate. Thus, understanding their priming mechanism is of importance for metabolic engineering. Insight into the loading process of anthranilate into the biosynthetic pathway for the quinoline alkaloids aurachins has been provided by the sequencing of a partial biosynthetic gene cluster in the myxobacterium Stigmatella aurantiaca.

Pyridoxal-5'-phosphate-dependent enzymes involved in biotin biosynthesis: structure, reaction mechanism and inhibition.

The four last steps of biotin biosynthesis, starting from pimeloyl-CoA, are conserved among all the biotin-producing microorganisms. Two enzymes of this pathway, the 8-amino-7-oxononanoate synthase (AONS) and the 7,8-diaminopelargonic acid aminotransferase (DAPA AT) are dependent on pyridoxal-5'-phosphate (PLP). This review summarizes our current understanding of the structure, reaction mechanism and inhibition on these two interesting enzymes.

The genome sequence of the cyanobacterium Oscillatoria sp. PCC 6506 reveals several gene clusters responsible for the biosynthesis of toxins and secondary metabolites.

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites.

Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis.

Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506.

In vitro reconstitution of the first steps of anatoxin-a biosynthesis in Oscillatoria PCC 6506: from free L-proline to acyl carrier protein bound dehydroproline.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillatoria PCC 6506.

Evidence that biosynthesis of the neurotoxic alkaloids anatoxin-a and homoanatoxin-a in the cyanobacterium Oscillatoria PCC 6506 occurs on a modular polyketide synthase initiated by L-proline.

Anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria such as Oscillatoria PCC 6506. Sequencing of the genome of this strain is underway, and we have identified a 29 kb DNA fragment containing a sequence called ks2 that we previously showed to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a. Bioinformatic analysis of this 29 kb fragment revealed a cluster of genes, which were annotated.

Identification of a polyketide synthase coding sequence specific for anatoxin-a-producing Oscillatoria cyanobacteria.

We report the identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase. Using 50 axenic cyanobacteria, we found this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, indicating its likely involvement in the biosynthesis of these toxins.

Inhibition of 7,8-diaminopelargonic acid aminotransferase from Mycobacterium tuberculosis by chiral and achiral anologs of its substrate: biological implications.

7,8-Diaminopelargonic acid aminotransferase (DAPA AT), a potential drug target in Mycobacterium tuberculosis, transforms 8-amino-7-oxononanoic acid (KAPA) into DAPA. We have designed an analytical method to measure the enantiomeric excess of KAPA, based on the derivatization of its amine function, by ortho-phtalaldehyde and N-acetyl-l-cysteine, followed by high pressure liquid chromatography separation. Using this methodology and enantiopure samples of KAPA it appeared that racemization of KAPA occurs rapidly (half-lives from 1 to 8 h) not only in 4 M HCl but more importantly in the usual pH range, from 7 to 9.

7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic target. Characterization and inhibition studies.

Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S-adenosyl-l-methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5'-phosphate-dependent enzymes and behaves as a homotetramer in solution.

Inhibition of thiamin diphosphate dependent enzymes by 3-deazathiamin diphosphate.

3-Deazathiamin diphosphate (deazaTPP) and a second thiamin diphosphate (TPP) analogue having a benzene ring in place of the thiazolium ring have been synthesised. These compounds are both extremely potent inhibitors of pyruvate decarboxylase from Zymomonas mobilis; binding is competitive with TPP and is essentially irreversible even though no covalent linkage is formed. DeazaTPP binds approximately seven-fold faster than TPP and at least 25,000-fold more tightly (K(i) less than 14 pM).

Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin.

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e.