New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a d ‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l ‐aculose or l ‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an l ‐aculose moiety to 3‐OH of the angucycline core.     

Biosynthetic Gene Cluster of a d‐Tryptophan‐Containing Lasso Peptide,MS‐271

MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271.     

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.     

Biosynthesis of Phenolic Glycosides from Phenylpropanoid and Benzenoid Precursors in <Emphasis Type="Italic">Populus</Emphasis>

Salicylate-containing phenolic glycosides (PGs) are abundant and often play a dominant role in plant-herbivore interactions of Populus and Salix species (family Salicaceae), but the biosynthetic pathway to PGs remains unclear. Cinnamic acid (CA) is thought to be a precursor of the salicyl moiety of PGs. However, the origin of the 6-hydroxy-2-cyclohexen-on-oyl (HCH) moiety found in certain PGs, such as salicortin, is not known. HCH is of interest because it confers toxicity and antifeedant properties against herbivores. We incubated Populus nigra leaf tissue with stable isotope-labeled CA, benzoates, and salicylates, and measured isotopic incorporation levels into both salicin, the simplest PG, and salicortin. Labeling of salicortin from [¹³C₆]-CA provided the first evidence that HCH, like the salicyl moiety, is a phenylpropanoid derivative. Benzoic acid and benzaldehyde also labeled both salicyl and HCH, while benzyl alcohol labeled only the salicyl moiety in salicortin. Co-administration of unlabeled benzoates with [¹³C₆]-CA confirmed their contribution to the biosynthesis of the salicyl but not the HCH moiety of salicortin. These data suggest that benzoate interconversions may modulate partitioning of phenylpropanoids to salicyl and HCH moieties, and hence toxicity of PGs. Surprisingly, labeled salicyl alcohol and salicylaldehyde were readily converted to salicin, but did not result in labeled salicortin. Co-administration of unlabeled salicylates with labeled CA suggested that salicyl alcohol and salicylaldehyde may have inhibited salicortin biosynthesis. A revised metabolic grid model of PG biosynthesis in Populus is proposed, providing a guide for functional genomic analysis of the PG biosynthetic pathway.     

Pneumocandin Biosynthesis: Involvement of a trans‐Selective Proline Hydroxylase

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans‐4‐ and trans‐3‐hydroxyproline, as well as trans‐3‐hydroxy‐4‐methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α‐ketoglutarate/Fe^II‐dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L ‐proline, GloF generates trans‐4‐ and trans‐3‐hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans‐4‐Methyl‐L ‐proline is converted into the corresponding trans‐3‐hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A₀ and B₀ in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far.     

New Aminocoumarins from the Rare Actinomycete Catenulispora acidiphila DSM 44928: Identification,Structure Elucidation,and Heterologous Production

Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L ^?1 in C. acidiphila to 60 mg L ^?1 in S. coelicolor M1152. By HR‐MS and NMR analysis, cacibiocin A was found to contain a 3‐amino‐4,7‐dihydroxycoumarin moiety linked by an amide bond to a pyrrole‐2,5‐dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6′ and 8′ of the aminocoumarin moiety.     

Biosynthesis of Nudicaulins: A 13CO2‐Pulse/Chase Labeling Study with Papaver nudicaule

Nudicaulins are unique alkaloids responsible for the yellow color of the petals of some papaveraceaous plants. To elucidate the unknown biosynthetic origin of the skeleton, a ¹³CO₂‐pulse/chase experiment was performed with growing Papaver nudicaule plants. ¹³C NMR analysis revealed more than 20 multiple ¹³C‐enriched isotopologues in nudicaulins from the petals of ¹³CO₂‐labeled plants. The complex labeling pattern was compared with the isotopologue composition of a kaempferol derivative that was isolated from petals of the same ¹³CO₂‐labeled plants. The deconvolution of the labeling profiles indicated that the nudicaulin scaffold is assembled from products or intermediates of indole metabolism, the phenylpropanoid pathway, and the polyketide biosynthesis. Naringenin‐type compounds and tryptophan/tryptamine are potential substrates for the condensation reaction finally generating the aglycone skeleton of nudicaulins.     

NAD+‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

The unique five‐membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d ‐glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar‐derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD⁺‐dependent alcohol dehydrogenase PctP and pyridoxal 5′‐phosphate dependent aminotransferase PctC converted N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophonone into 3′‐amino‐3′‐deoxy‐N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophenone. Further, N‐acetyl‐d ‐glucosaminyl‐3‐aminophenyl‐β‐oxopropanoic acid ethyl ester was converted into the corresponding 3′‐amino derivative. However, PctP did not oxidize most of the tested d ‐glucose derivatives, including UDP‐GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.     

Synthesis of trisubstituted C18 pyrrole fatty ester derivatives

Reaction of methyl 10(11)-dicarbethoxymethyl-9,12-dioxooctadecanoate (1a,1b) with ammonium acetate furnished a mixture of positional isomers of a pyrrole derivative, methyl 9,12-imino-10(11)-dicarbethoxymethyl-9,11-octadecadienoate (2a,2b). Decarboxylation of the mixture of compounds 2a,2b with sodium carbonate in aqueous methanol yielded a mixture of compounds 3a,3b containing a CH₂COOCH₃ group at the 3- or 4-position of the pyrrole ring after esterification. Heating of the hydrolyzed mixture of compounds 3a,3b at 180°C for 1 h gave the desired trisubstituted pyrrole derivatives, methyl 9,12-imino-10(11)-methyl-9,11-octadecadienoate (4a,4b), containing a methyl group at the 3- or 4-position of the pyrrole nucleus. The structures of the products and intermediates were confirmed by infrared, and by¹H and¹³C nuclear magnetic resonance spectroscopy.     

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

Key Residues in Octyl‐Tridecaptin A1 Analogues Linked to Stable Secondary Structures in the Membrane

Tridecaptin A₁ is a linear antimicrobial lipopeptide comprised of 13 amino acids, including three diaminobutyric acid (Dab) residues. It displays potent activity against Gram‐negative bacteria, including multidrug‐resistant strains. Using solid‐phase peptide synthesis, we performed an alanine scan of a fully active analogue, octyl‐tridecaptin A₁, to determine key residues responsible for activity. The synthetic analogues were tested against ten organisms, both Gram‐positive and Gram‐negative bacteria. Modification of D ‐Dab8 abolished activity, and marked decreases were observed with substitution of D ‐allo‐Ile12 and D ‐Trp5. Circular dichroism showed that octyl‐tridecaptin A₁ adopts a secondary structure in the presence of model phospholipid membranes, which was weakened by D ‐Dab8‐D ‐Ala, D ‐allo‐Ile12‐D ‐Ala, and D ‐Trp5‐D ‐Ala substitutions. The antimicrobial activity of the analogues is directly correlated to their ability to adopt a stable secondary structure in a membrane environment.     

Spectroelectrochemistry of pyrrole oligomers in the presence of acrylamide

Soluble pyrrole oligomers were characterized during electropolymerization of pyrrole in the presence of acrylamide (in acetonitrile) which stabilizes radical cations of pyrrole allowing the reaction to be followed by spectroscopic and spectroelectrochemical means. The role of the applied electrical conditions and the effect of the presence of acrylamide on the formation of pyrrole oligomers in solution, and on the resulting polymer on an electrode surface (both Pt and indium tin oxide), were investigated. The soluble and insoluble products were characterized using UV‐visible and FTIR spectroscopy, cyclic voltammetry and a four‐point probe technique. Polypyrrole and poly(pyrrole–acrylamide) free‐standing films had conductivity values of about 90 and 1 S cm^?1, respectively. Spectroscopic, cyclovoltammetric and conductivity results support the incorporation of acrylamide into intermediate species that may have useful application in industry. © 2002 Society of Chemical Industry     

Analysis of the kinetics and current efficiency of pyrrole galvanostatic electropolymerization

We propose a modified kinetic equation for the galvanostatic electropolymerization of pyrrole based on equal rates of monomer disappearance and its galvanostatic electropolymerization associated with applied current (I). The equation is distinguished by a zero‐order kinetic plot and takes into account the effects of the pyrrole initial concentration ([M]₀) and current efficiency (η). We propose a mechanism for obtaining a η of less than 100% and increasing η with increasing [M]₀ and I on the basis of the diffusion of radical cations (M^?+) from the anode surface to the bulk solution after the electroreduction of M^?+ to monomer molecules at the cathode. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 1167–1169, 2005     

Sphinganine‐Like Biogenesis of (E)‐1‐Nitropentadec‐1‐ene in Termite Soldiers of the Genus Prorhinotermes

In 1974, (E)‐1‐nitropentadec‐1‐ene, a strong lipophilic contact poison of soldiers of the termite genus Prorhinotermes, was the first‐described insect‐produced nitro compound. However, its biosynthesis remained unknown. In the present study, we tested the hypothesis that (E)‐1‐nitropentadec‐1‐ene biosynthesis originates with condensation of amino acids with tetradecanoic acid. By using in vivo experiments with radiolabeled and deuterium‐labeled putative precursors, we show that (E)‐1‐nitropentadec‐1‐ene is synthesized by the soldiers from glycine or L ‐serine and tetradecanoic acid. We propose and discuss three possible biosynthetic pathways.     

Novel hyperbranched molecules containing pyrrole units from diacetylene compounds

Novel hyperbranched molecules containing pyrrole units were obtained from ortho-, meta-, and para-diaminodiphenyldiacetylenes, as AB₂ type monomers by one-step polymerization. Diacetylenic fragments reacted with terminal amino groups in the presence of copper chloride to give pyrrole units. Diaminodiphenyldiacetylene monomers have been synthesized from ethynilanilines in three steps. The novel monomers and hyperbranched molecules were characterized by NMR, IR and thermal analysis.     

Biochemical Investigations of Two 6‐DMATS Enzymes from Streptomyces Reveal New Features of L‐Tryptophan Prenyltransferases

Two putative prenyltransferase genes, SAML0654 and Strvi8510, were identified in Streptomyces ambofaciens and Streptomyces violaceusniger, respectively. Their deduced products share 63 % sequence identity. Biochemical investigations with recombinant proteins demonstrated that L ‐tryptophan and derivatives, including D ‐tryptophan, 4‐, 5‐, 6‐ and 7‐methyl‐dl ‐tryptophan, were well accepted by both enzymes in the presence of DMAPP. Structural elucidation of the isolated products revealed regiospecific prenylation at C‐6 of the indole ring and proved unequivocally the identification of two very similar 6‐dimethylallyltryptophan synthases (6‐DMATS). Detailed biochemical investigations with SAML0654 proved L ‐tryptophan to be the best substrate (K_m 18 μm, turnover 0.3 s^?1). Incubation with different prenyl donors showed that they also accepted GPP and catalyzed the same specific prenylation. Utilizing GPP as a prenyl donor has not been reported for tryptophan prenyltransferases previously. Both enzymes also catalyzed prenylation of some hydroxynaphthalenes; this has not previously been described for bacterial indole prenyltransferases. Interestingly, SAML0654 transferred prenyl moieties onto the unsubstituted ring of hydroxynaphthalenes.     

Novel Aeruginosin‐865 from Nostoc sp. as a Potent Anti‐inflammatory Agent

Aeruginosin‐865 (Aer‐865), isolated from terrestrial cyanobacterium Nostoc sp. Luke?ová 30/93, is the first aeruginosin‐type peptide containing both a fatty acid and a carbohydrate moiety, and is the first aeruginosin to be found in the genus Nostoc. Mass spectrometry, chemical and spectroscopic analysis as well as one‐ and two‐dimensional NMR and chiral HPLC analysis of Marfey derivatives were applied to determine the peptidic sequence: D ‐Hpla, D ‐Leu, 5‐OH‐Choi, Agma, with hexanoic and mannopyranosyl uronic acid moieties linked to Choi. We used an AlphaLISA assay to measure the levels of proinflammatory mediators IL‐8 and ICAM‐1 in hTNF‐α‐stimulated HLMVECs. Aer‐865 showed significant reduction of both: with EC₅₀ values of (3.5±1.5) μg mL^?1 ((4.0±1.7) μM ) and (50.0±13.4) μg mL^?1 ((57.8±15.5) μM ), respectively. Confocal laser scanning microscopy revealed that the anti‐inflammatory effect of Aer‐865 was directly associated with inhibition of NF‐κB translocation to the nucleus. Moreover, Aer‐865 did not show any cytotoxic effect.     

An Iterative O‐Methyltransferase Catalyzes 1,11‐Dimethylation of Aspergillus fumigatus Fumaric Acid Amides

S‐adenosyl‐l ‐methionine (SAM)‐dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11‐dimethyl esters of fumaryl‐l ‐tyrosine and fumaryl‐l ‐phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size‐exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.     

Synthesis, characterization and electrochromic properties of a conducting copolymer of pyrrole functionalized polystyrene with pyrrole

A well-defined polystyrene (PSt) based polymer containing at one end-chain 3,5-dibromobenzene moiety, prepared by atom transfer radical polymerization (ATRP), was modified in two reaction steps. First one constitutes a Suzuki coupling reaction between aromatic dibromine functional polymer and 3-aminophenylboronic acid, when a diamino-containing intermediate was obtained. The second step is a condensation reaction between the diamino functional polystyrene and 2-pyrrole aldehyde. Thus, a polymer containing a conjugated sequence having pyrollyl groups at the extremities was synthesized. The presence of oxidable pyrrole groups in the structure of the polymer permitted further electropolymerization. The structures of intermediate polymers were analyzed by spectral methods (¹H NMR, FTIR). Electrochemical copolymerization of pyrrole functionalized polymer (PStPy) with pyrrole was carried out in acetonitrile (ACN)-tetrabutylammonium tetrafluoroborate (TBAFB) solvent electrolyte couple. Characterization of the resulting copolymer were performed via Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), spectroelectrochemical analysis, and kinetic study. Spectroelectrochemical analysis show that the copolymer of PStPy with Py has an electronic band gap (due to π-π* transition) of 2.4 eV at 393 nm, with a yellow color in the fully reduced form and a blue color in the fully oxidized form. Via kinetic studies, the optical contrast %ΔT was found to be 20% for P(PStPy-co-Py). Results showed that the time required to reach 95% of the ultimate T was 1.7 s for the P(PStPy-co-Py).