Para-position derivatives of fungal anthelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes

PF1022A, a cyclooctadepsipeptide possessing strong anthelmintic properties and produced by the filamentous fungus Rosellinia sp. PF1022, consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate. PF1022A derivatives obtained through modification of their benzene ring at the para-position with nitro or amino groups act as valuable starting materials for the synthesis of compounds with improved anthelmintic activities. Here we describe the production of such derivatives by fermentation through metabolic engineering of the PF1022A biosynthetic pathway in Rosellinia sp. PF1022. Three genes cloned from Streptomyces venezuelae, and required for the biosynthesis of p-aminophenylpyruvate from chorismate in the chloramphenicol biosynthetic pathway, were expressed in a chorismate mutase-deficient strain derived from Rosellinia sp. PF1022. Liquid chromatography-mass spectrometry and NMR analyses confirmed that this approach facilitated the production of PF1022A derivatives specifically modified at the para-position. This fermentation method is environmentally safe and can be used for the industrial scale production of PF1022A derivatives.     

Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi

The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures was measured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase of cultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present during growth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was present at every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is a constitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand in contrast to the common assumption that production of secondary metabolites underlies regulatory control, leading to separation of the trophophase and the idiophase.     

Structure-Activity Relationship of Anthelmintic Cyclooctadepsipeptides

The relationship between cyclooctadepsipeptides and their anthelmintic efficacy was examined by converting the natural products, PF1022A, PF1022E and PF1022H. Some analogues substituted at the para position of the phenyllactate moiety showed higher or equivalent activity against the parasitic nematode, Ascaridia galli in chicken when compared with the parent compounds. It is suggested that lipophilicity and the polar surface area, in addition to structural requirements of the derivatives, influenced the anthelmintic efficacy in vivo.     

Structure-activity relationship of anthelmintic cyclooctadepsipeptides

The relationship between cyclooctadepsipeptides and their anthelmintic efficacy was examined by converting the natural products, PF1022A, PF1022E and PF1022H. Some analogues substituted at the para position of the phenyllactate moiety showed higher or equivalent activity against the parasitic nematode, Ascaridia galli in chicken when compared with the parent compounds. It is suggested that lipophilicity and the polar surface area, in addition to structural requirements of the derivatives, influenced the anthelmintic efficacy in vivo.     

Anthelmintic cyclcooctadepsipeptides: complex in structure and mode of action

The broad-spectrum anthelmintic cyclooctadepsipeptide PF1022A is a fungal metabolite from Rosellinia sp. PF1022, which is a Mycelia sterilia found on the leaves of Camellia japonica. A broad range of structurally related cyclooctadepsipeptides has been characterized and tested for anthelmintic activities. These metabolites have been used as starting points to generate semisynthetic derivatives with varying nematocidal capacity. Predominant among these compounds is emodepside, which exhibits a broad nematocidal potential against gastrointestinal and extraintestinal parasites. Here we review the chemical biology and mode of action of cyclooctadepsides with particular attention to PF1022A and emodepside. We illustrate how they target nematode neuromuscular function, opening up new avenues for antiparasitic treatments with potential capability for important selective toxicity.     

Selective synthesis of depsipeptides by the immobilized multienzyme enniatin synthetase

Summary The multifunctional enzyme enniatin synthetase was immobilized by adsorption to propyl agarose. The immobilized multienzyme retained 45% of the activity of the free enzyme; an operational half-life of about 15 h was estimated. Selective synthesis of several different enniatin homologues was achieved with propyl agarose-bound enniatin synthetase. In addition to enniatin A, B, and C formation, a selective synthesis of non-naturally occurring depsipeptides, containing norvaline, norleucine, or -aminobutyric acid as sole amino acid moieties, was observed.     

Use of network analysis of metabolic systems in bioengineering

Basic ideas and recent developments in network analysis of metabolic systems and various applications of this analysis in bioengineering are reviewed. Central concepts are the null-space to the stoichiometry matrix and the elementary flux modes. The applicability of elementary-modes analysis in biotechnology is illustrated by the synthesis of the cyclooctadepsipeptides PF1022 in the fungus Mycelia sterilia. Network analysis is also useful in metabolic flux analysis. In particular, a procedure for finding out which reaction rates can be uniquely calculated in underdetermined reaction networks is outlined. The concept of 'enzyme subsets' is explained and its use for analysing genetic regulation is demonstrated. In particular, the correlation between expression data concerning the diauxic shift in yeast and the enzyme subsets in yeast metabolism is discussed.     

Decreased emodepside sensitivity in unc-49 γ-aminobutyric acid (GABA)-receptor-deficient Caenorhabditis elegans

Emodepside, a semi-synthetic derivative of PF1022A, belongs to a new class of anthelmintic drugs, the cyclooctadepsipeptides, and shows good efficacy against macrocyclic lactone-, levamisole- or benzimidazole-resistant nematode populations. Although putative receptors for emodepside have already been discovered, its mode of action is still not fully understood. The involvement of the γ-aminobutyric acid (GABA)-receptor on the PF1022A mode of action has previously been postulated. Therefore, a possible role of the GABA-receptor, unc-49, in the mode of action of emodepside was investigated using two different Caenorhabditis elegans in vitro assays, a motility assay and a development assay. It was found that there is a clearly reduced sensitivity against emodepside of strains carrying a GABA-receptor, unc-49, loss of function mutation compared with N2 wild type C. elegans. To transfer these results from the model system to parasitic nematodes, the Toxocara canis unc-49B cDNA sequence was identified and used in a rescue experiment. The emodepside-susceptible phenotype could be fully rescued by injection of the T. canis unc-49B cDNA sequence. We believe that this is the first functional rescue of a C. elegans mutant strain with a gene from a clade III parasitic nematode. These findings, together with the earlier data on GABA-receptor binding of PF1022A, suggest that the GABA(A)-receptor UNC-49 is associated with the emodepside mode of action. However, the only partially resistant phenotype of the loss of function mutants indicates that other pathways play a more significant role.     

Efficacy of Cyclooctadepsipeptides and Aminophenylamidines against Larval,Immature and Mature Adult Stages of a Parasitologically Characterized Trichurosis Model in Mice

Background

The genus Trichuris includes parasites of major relevance in veterinary and human medicine. Despite serious economic losses and enormous impact on public health, treatment options against whipworms are very limited. Additionally, there is an obvious lack of appropriately characterized experimental infection models. Therefore, a detailed parasitological characterization of a Trichuris muris isolate was performed in C57BL/10 mice. Subsequently, the in vivo efficacies of the aminophenylamidines amidantel, deacylated amidantel (dAMD) and tribendimidine as well as the cyclooctadepsipeptides emodepside and in particular PF1022A were analyzed. This was performed using various administration routes and treatment schemes targeting histotropic and further developed larval as well as immature and mature adult stages.

Methodology/Principal Findings

Duration of prepatent period, time-dependent localization of larvae during period of prepatency as well as the duration of patency of the infection were determined before drugs were tested in the characterized trichurosis model. Amidantel showed no effect against mature adult T. muris. Tribendimidine showed significantly higher potency than dAMD after oral treatments (ED₅₀ values of 6.5 vs. 15.1 mg/kg). However, the opposite was found for intraperitoneal treatments (ED₅₀ values of 15.3 vs. 8.3 mg/kg). When emodepside and PF1022A were compared, the latter was significantly less effective against mature adults following intraperitoneal (ED₅₀ values of 6.1 vs. 55.7 mg/kg) or subcutaneous (ED₅₀ values of 15.2 vs. 225.7 mg/kg) administration. Only minimal differences were observed following oral administration (ED₅₀ values of 2.7 vs. 5.2 mg/kg). Triple and most single oral doses with moderate to high dosages of PF1022A showed complete efficacy against histotropic second stage larvae (3×100 mg/kg or 1×250 mg/kg), further developed larvae (3×10 mg/kg or 1×100 mg/kg) and immature adults (3×10 mg/kg or 1×100 mg/kg). Histotropic first stage larvae were only eliminated after three doses of PF1022A (3×100 mg/kg) but not after a single dose.

Conclusions/Significance

These results indicate that the cyclooctadepsipeptides are a drug class with promising candidates for further evaluation for the treatment of trichurosis of humans and livestock animals in single dose regimens.     

Enniatin Production by Fusarium Strains and Its Effect on Potato Tuber Tissue

Several Fusarium strains produce the cyclohexadepsipeptide enniatin, a host-nonspecific phytotoxin. Enniatins are synthesized by the 347-kDa multifunctional enzyme enniatin synthetase. In the present study, 36 Fusarium strains derived from a wide range of host plants were characterized with respect to enniatin production in different media. Thirteen of these strains produced enniatins on one or more of these media. To determine whether enniatin production affected virulence, an assay on potato tuber tissue was performed. Seven enniatin-producing and 16 nonproducing strains induced necrosis of potato tuber tissue, so that enniatin synthesis is not essential for the infection of potato tuber tissue. The application of a mixture of enniatins to slices of potato tuber, however, caused necrosis of the tissue. Therefore, enniatin production by the enniatin-synthesizing strains may affect their pathogenicity. The enniatin synthetase gene (esyn1) of Fusarium scirpi ETH 1536 was used as a probe to determine if similar sequences were present in the strains examined. In Southern blot analyses, DNA sequences hybridizing with the esyn1 probe were present in all but two of the strains examined. In some cases, enniatin-nonproducing strains had the same hybridization pattern as enniatin producers.     

Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described

Cyclosporin A and its homologues are synthesized by a single multifunctional enzyme from their precursor amino acids. Cyclosporin synthetase is a polypeptide chain with a molecular mass of approximately 800 kDa. In 3% polyacrylamide-sodium dodecyl sulfate gels it shows a single band of approximately 650 kDa, which appears to not be glycosylated. The enzyme could be purified to near-homogeneity in five steps. A 72-fold purification was obtained. All constitutive amino acids of cyclosporins are activated as thioesters via aminoadenylation by the same enzyme. Then N-methylation of the thioester-bound amino acids which are present in methylated form in the cyclosporin molecule takes place, whereby S-adenosyl-L-methionine serves as the methyl group donor. Methyltransferase activity is an integral entity of the enzyme; this could be shown by a photoaffinity labeling method. 4'-Phosphopantetheine is a prosthetic group of cyclosporin synthetase similar to other peptide and depsipeptide synthetases. Cyclosporin synthetase shows cross-reactions with monoclonal antibodies directed against enniatin synthetase.     

Mechanism of depsipeptide formation catalyzed by enniatin synthetase

Covalently bound intermediates of enniatin B synthesis could be isolated from enniatin synthetase by treatment with performic acid. By comparison with products of mild alkaline cleavage of authentic enniatin B they could be identified as the dipeptide D-2-hydroxyisovaleryl-N-methylvaline and the corresponding tetrapeptide. Synthesis of enniatins apparently proceeds via condensation of dipeptides. This was confirmed by the use of the substrate analogue isovaleric acid, which has shown to be a strong inhibitor for enniatin synthesis by formation of N-isovaleryl-N-methyl valine.     

The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit

Barbamide was extracted from the marine cyanobacterium Lyngbya majuscula strain 19L as a chlorinated lipopeptide for its potent molluscicidal activity. Precursor incorporation studies indicated that it is derived from acetate, L-phenylalanine, L-leucine and L-cysteine. The gene cluster responsible for biosynthesis of barbamide (bar) was cloned and characterized in this study. DNA sequence analysis of cosmid pLM49 revealed a cluster of 12 open reading frames (barA-barK) extending 26 kb including the expected polyketide synthase and non-ribosomal peptide synthetase modules and tailoring genes. The genetic architecture and domain organization of the bar cluster supports the assignment based on the apparent co-linearity of the systems. The activity assay of adenylation domains of barD (A(D)), barE (A(E)) and barG (A(G2) for module 2) in an amino acid-dependent ATP-pyrophosphate exchange experiment supports the conclusion that barbamide is synthesized from acetate, L-phenylalanine, L-cysteine and L-leucine with trichloroleucine as a direct precursor by a mixed polyketide synthase/non-ribosomal polypeptide synthetase. Assembly of barbamide includes unique biochemical mechanisms for chlorination, one-carbon truncation during chain elongation, E-double bond formation and thiazole ring formation.     

Cysteinyl-tRNA synthetase from Bacillus stearothermophilus. A structural and functional monomer.

A procedure is described for the purification of cysteinyl-tRNA synthetase as a side product of a multi-enzyme isolation from Bacillus stearothermophilus. The native and denatured enzyme are both shown to have a molecular weight of 54000 by gel filtration and sodium dodecyl sulphate/polyacrylamide gel electrophoresis respectively. Fingerprinting and peptide counting indicate that the polypeptide chain has a nonrepeating primary structure. The enzyme has only one binding site for each of its substrates (cysteine, ATP and tRNACys) as judged by equilibrium dialysis, active-site titration and fluorescence quenching. No evidence for the dimerisation of the enzyme in the presence of these substrates could be found. We conclude that cysteinyl-tRNA synthetase, which is the smallest aminoacyl-tRNA synthetase yet described, is both structurally and functionally monomeric.     

The effects of phospholipids on the activation of glutamate dehydrogenase by L-leucine.

Glutamate dehydrogenase in disrupted mitochondrial preparations is activated by L-leucine to a much greater extent than is the purified enzyme. A factor, or factors, responsible for modulating the sensitivity of L-leucine is lost during the purification of the enzyme. Although both cardiolipin and phosphatidylserine are inhibitors of the enzyme, only the inhibition by the former phospholipid is reversed by L-leucine. The inhibition of glutamate dehydrogenase by its binding to cardiolipin in the disrupted mitochondrial preparations and its relief by L-leucine could account for the greater sensitivity of such preparations to activation by that amino acid.     

Enniatin synthetase is a monomer with extended structure: evidence for an intramolecular reaction mechanism

Enniatin synthetase (Esyn), a 347-kDa multienzyme consisting of two substrate activation modules, is responsible for the nonribosomal formation of the cyclohexadepsipeptide enniatin. The synthesis follows the so-called thiol template mechanism. While this process is basically well established, no substantial insight into the 3-dimensional arrangement of these enzymes and possible interactions between them exists to date. To find out whether enniatin synthesis is an intramolecular process or the result of three interacting Esyn molecules (intermolecular), analytical ultracentrifugation equilibration studies were carried out. The molecular mass of Esyn was determined by ultracentrifugation and is in good agreement with that calculated from the ORF of the encoding gene, indicating that Esyn exists in solution as a monomer. This strongly suggests that synthesis of the cyclohexadepsipeptide enniatin follows an intramolecular reaction mechanism in which all three reaction cycles are catalyzed by a single Esyn molecule. This finding was supported by in vitro complementation studies in which [(14)C]-methylvalyl Esyn, upon incubation with the second substrate D-2-hydroxyisovaleric acid (D-Hiv) and ATP, did not yield radioactive enniatin. This confirms our previous assumption of an iterative reaction mechanism similar to that for fatty acid synthase. Furthermore, the sedimentation rate constant evaluated from analytical ultracentrifugation was lower (S(20,w)=14.1S) than expected (S(20,w)=16.9S) for a globular protein, indicating that Esyn has an extended structure.     

A novel family of bacterial dioxygenases that catalyse the hydroxylation of free L-amino acids

L-isoleucine-4-hydroxylase (IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. L-methionine, L-leucine, L-isoleucine and L-threonine were found to be catalysed by the investigated enzymes, producing L-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free L-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed.     

Efficacy of the cyclooctadepsipeptide PF1022A against Heligmosomoides bakeri in vitro and in vivo

The cyclooctadepsipeptide PF1022A derived from the fungus, Mycelia sterilia, is characterized by a broad spectrum of activity against different parasitic gastrointestinal nematodes of livestock. In the present work the anthelmintic activity of PF1022A against Heligmosomoides bakeri, a widely used laboratory model was studied. Albendazole, ivermectin and levamisole served as reference. In vitro, PF1022A showed low activity on embryonation but significantly inhibited egg hatch (10 and 100 μg/ml), whereas albendazole (10 and 100 μg/ml) revealed statistically significant inhibitions of both embryonation and egg hatch. PF1022A (1-100 μg/ml) completely inhibited larval movement at most examination points. Comparable significant anthelmintic activity on the larval stages of H. bakeri was observed with levamisole (48-100%), while slightly lower activities were observed with ivermectin (20-92%) and albendazole (0-87%) at 1-100 μg/ml. PF1022A and levamisole significantly inhibited motility and egg release of adult worms, while albendazole and ivermectin failed to demonstrate activity. Significant worm burden reductions were achieved with PF1022A, levamisole and ivermectin in vivo. For example, at 0·125 mg/kg PF1022A a worm burden reduction of 91·8% was observed. The use of drug combinations did not further enhance the in vitro and in vivo activity of PF1022A. In conclusion, further investigations are warranted with PF1022A, as the drug is characterized by significant larvicidal and nematocidal activity in vitro and in vivo.     

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.