naphthoquinones and flaviolin

The novel SARS-CoV-2 is the etiological agent causing the Coronavirus disease 2019 (COVID-19), which continues to become an inevitable pandemic outbreak. Over a short span of time, the structures of therapeutic target proteins for SARS-CoV-2 were identified based on the homology modelled structure of similar virus, SARS-CoV that transmitted rapidly in 2003. Since the outset of the disease, the research community has been looking for a potential drug lead. Out of all the known resolved structures related to SARS-CoV-2; 3-chymotrypsin (3 C) like protease (3CL

Topics: COVID-19; Fungi; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Naphthoquinones; Protease Inhibitors; SARS-CoV-2

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes.

Topics: Gene Deletion; Multigene Family; Naphthoquinones; Saccharopolyspora

Although the mechanisms of regulating secondary metabolism by LaeA remains unclear, the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation. In our previous sutdy, RNA-seq data has showed that the transcriptional level of many SM backbone genes could be upregulated by overexpressing LaeA. Herein, we analyzed the chemical profile of activated secondary metabolites in the variant of A. niger FGSC A1279 by overexpressing LaeA (OElaeA). 14 compounds were activated in A. niger FGSC A1279 OElaeA variant in the WATM medium. Chemical workup of organic extracts of the culture broth from the A. niger OElaeA mutant identified three pure compounds, flaviolin, orlandin and kotanin. The structures of these compounds were confirmed by HR-ESIMS, 1D/2D NMR, and computer assisted structure elucidation (CASE). Based on homologous alignment and comparison of literatures, the biosynthetic gene cluster (fla) of flaviolin was identified. The in vivo function of the backbone gene, flaA, encoding a multidomain non-reducing polyketide synthase (SAT-KS-AT-PT-ACP), was verified via gene knockout and chemical analysis. Finally, a biosynthetic model for fungal flaviolin was proposed.

Topics: Aspergillus niger; Coumarins; Fungal Proteins; Gene Expression Regulation, Fungal; Mass Spectrometry; Multigene Family; Naphthoquinones; Secondary Metabolism; Umbelliferones

The cross-coupling reaction is one of the most important chemical reactions in the modern organic chemistry. Biocatalysts capable of catalyzing C-C coupling reactions are desired in the chemical industry for sustainable development. Cytochrome P450 monooxygenases (P450s) have received considerable attention as biocatalysts capable of catalyzing such reactions. Here, we focused on actinomycete P450s, which have high homology with CYP158A2, involved in the oxidative C-C coupling reaction for flaviolin dimerization in Streptomyces coelicolor A3(2). The screening of a chemical library composed of 426 aromatic compounds identified several combinations of P450s and their potential substrates. The type-I difference spectrum indicated that the identified substrates bind to the active sites of a P450, named StVI from Streptomyces violaceusniger. A redshift of the absorption maximum of the reaction products, together with LC-MS analysis suggested the presence of extended conjugate systems in the products through direct C-C coupling between two aromatic rings. The results demonstrated that actinomycete P450s have great potential to be utilized as biocatalysts for oxidative C-C coupling reactions and to facilitate the synthesis of diverse coupling products.

Topics: Actinobacteria; Bacterial Proteins; Catalysis; Catalytic Domain; Cytochrome P-450 Enzyme System; Dimerization; Naphthoquinones; Oxidation-Reduction; Streptomyces coelicolor

Heterologous hosts are important platforms for engineering natural product biosynthesis. Escherichia coli is such a host widely used for expression of various biosynthetic enzymes. While numerous studies have been focused on optimizing the expression conditions for desired functional proteins, this work describes how supplement of exogenous nutrients into the fermentation broth influences the formation of natural products in E. coli. A type III polyketide synthase gene stts from Streptomyces toxytricini NRRL 15443 was heterogeneously expressed in E. coli BL21(DE3). This enzyme uses five units of malonyl-CoA to generate a polyketide 1,3,6,8-tetrahydroxynaphthalene, which can be spontaneously oxidized into a red compound flaviolin. In this work, we manipulated the fermentation broth of E. coli BL21(DE3)/pET28a-stts by supplying different nutrients including glucose and sodium pyruvate at different concentrations, from which six flaviolin derivatives 1-6 were produced. While addition of glucose yielded the production of 1-4, supplement of sodium pyruvate into the induced broth of E. coli BL21(DE3)/pET28a-stts resulted in the synthesis of 5 and 6, suggesting that different nutrients may enable E. coli to generate different metabolites. These products were purified and structurally characterized based on the spectral data, among which 2-6 are novel compounds. These molecules were formed through addition of different moieties such as acetone and indole to the flaviolin scaffold. The concentrations of glucose and sodium pyruvate and incubation time affect the product profiles. This work demonstrates that supplement of nutrients can link certain intracellular metabolites to the engineered biosynthetic pathway to yield new products. It provides a new approach to biosynthesizing novel molecules in the commonly used heterologous host E. coli.

Topics: Acyltransferases; Biosynthetic Pathways; Culture Media; Escherichia coli; Fermentation; Glucose; Malonyl Coenzyme A; Naphthols; Naphthoquinones; Polyketides; Pyruvic Acid; Streptomyces

Recent advances in genome sequencing, combined with bioinformatic analysis, has led to the identification of numerous novel natural product gene clusters, particularly in actinomycetes of terrestrial and marine origin. Many of these gene clusters encode uncharacterised Type III polyketide synthases. To facilitate the study of these genes and their potentially novel products, we set out to construct an actinomycete expression host specifically designed for the heterologous expression of Type III PKS genes and their gene clusters.. A derivative of Streptomyces coelicolor A3(2) designed for the expression of Type III polyketide synthase (PKS) genes was constructed from the previously engineered expression strain S. coelicolor M1152 [Δact Δred Δcpk Δcda rpoB(C1298T)] by removal of all three of the endogenous Type III PKS genes (gcs, srsA, rppA) by PCR targeting. The resulting septuple deletion mutant, M1317, proved to be an effective surrogate host for the expression of actinobacterial Type III PKS genes: expression of the reintroduced gcs gene from S. coelicolor and of the heterologous rppA gene from Streptomyces venezuelae under the control of the constitutive ermE* promoter resulted in copious production of germicidin and flaviolin, respectively.. The newly constructed expression host S. coelicolor M1317 should be particularly useful for the discovery and analysis of new Type III polyketide metabolites.

Topics: Bioreactors; Genetic Engineering; Multigene Family; Mutagenesis, Site-Directed; Naphthoquinones; Organisms, Genetically Modified; Polyketide Synthases; Pyrones; Streptomyces coelicolor

A type III polyketide biosynthetic gene cluster has been discovered in the industrially important strain Streptomyces toxytricini NRRL 15443, including four genes stp450-1, stts, stp450-2, and stmo. The stts gene encodes a putative type III polyketide synthase that is homologous to RppA, a 1,3,6,8-tetrahydroxynaphthalene (THN) synthase from Streptomyces griseus. The deduced protein product of stmo resembles the cupin-containing monooxygenase MomA from Streptomyces antibioticus that oxidizes THN into flaviolin. Two cytochrome P450s (CYPs), StP450-1 and StP450-2, are present in the gene cluster. StTS was overexpressed in Escherichia coli BL21(DE3) and identified as a THN synthase. The synthesized THN can be easily oxidized into flaviolin by air. Both CYPs were reconstituted in E. coli BL21(DE3) and can oxidize flaviolin to form oligomers. The k(cat)/K(m) values for StP450-1 and StP450-2 were 0.28 and 0.71 min⁻¹ mM⁻¹, respectively. UV irradiation test showed that expression of StTS in E. coli BL21(DE3) significantly protects the cells from UV radiation, and coexpression of StTS and StP450-1 provides even stronger protection.

Topics: Acyltransferases; Amino Acid Sequence; Bacterial Proteins; Cloning, Molecular; Cytochrome P-450 Enzyme System; Escherichia coli; Gene Expression; Isoenzymes; Mixed Function Oxygenases; Molecular Sequence Data; Multigene Family; Naphthols; Naphthoquinones; Oxidation-Reduction; Plasmids; Radiation-Protective Agents; Sequence Alignment; Streptomyces; Streptomyces griseus; Ultraviolet Rays

Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/PactI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.

Topics: Acyltransferases; Gene Expression; Genes, Reporter; Naphthols; Naphthoquinones; Oxidation-Reduction; Recombinant Proteins; Saccharopolyspora; Spectrophotometry; Streptomyces

The napyradiomycins are an intriguing family of halogenated natural products with activity against several tumor cell lines as well as some of the worst bacterial strains known to humanity, including methicillin-resistant Staphylococcus aureas and vancomycin-resistant strains of Enterococcus faecium. This communication delineates the first asymmetric total synthesis of (-)-napyradiomycin A1 by a strategy that features a two-step total synthesis of flaviolin, the first highly asymmetric halogenation of a simple alkene, and a Johnson-Claisen rearrangement that generates a quaternary carbon next to a glucal-like oxygen.

Topics: Alkenes; Halogenation; Isomerism; Naphthoquinones

We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA).

Topics: Acyltransferases; Amino Acid Sequence; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Naphthols; Naphthoquinones; Phenotype; Sequence Alignment; Sequence Analysis, Protein; Spectrometry, Mass, Electrospray Ionization; Streptomyces

Cytochrome P450 158A2 (CYP158A2) has been shown to catalyze an unusual oxidative C-C coupling reaction to polymerize flaviolin and form highly conjugated pigments (three isomers of biflaviolin and one triflaviolin) in Streptomyces coelicolor A3(2) which protect the soil bacterium from deleterious effects of UV irradiation (Zhao B. et al. (2005) J. Biol. Chem. 280, 11599-11607). The present studies demonstrate that the subfamily partner CYP158A1, sharing 61% amino acid identity with CYP158A2, can also catalyze the same flaviolin dimerization reactions, but it generates just two of the three isomers of biflaviolin that CYP158A2 produces. Furthermore, the two CYP158A1 products have very different molar ratios compared with the corresponding CYP158A2 products, indicating that each enzyme maintains its own stereo- and regiospecificity. To find an explanation for these differences, three CYP158A1 structures have been solved by X-ray crystallography and have been compared with those for CYP158A2. The structures reveal surprising differences. Particularly, only one flaviolin molecule is present close to the heme iron in CYP158A1, and the second flaviolin molecule binds at the entrance of the putative substrate access channel on the protein distal surface 9 A away. Our work describes two members of the same P450 subfamily, which produce the same products by oxidative C-C coupling yet show very different structural orientations of substrate molecules in the active site.

Topics: Bacterial Proteins; Binding Sites; Carbon-Carbon Double Bond Isomerases; Crystallography, X-Ray; Cytochrome P-450 Enzyme System; Dimerization; Models, Molecular; Naphthoquinones; Oxidation-Reduction; Pigments, Biological; Protein Conformation; Streptomyces coelicolor; Substrate Specificity

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.

Topics: Acyltransferases; Base Sequence; Myxococcales; Naphthoquinones; Phylogeny; Pseudomonas

Cytochrome P450 158A2 (CYP158A2) can polymerize flaviolin to red-brown pigments, which may afford physical protection to the organism, possibly against the deleterious effects of UV radiation. We have found that the small molecule malonic acid enables cocrystallization of this mixed function oxidase with the azole inhibitor 4-phenylimidazole. The presence of malonate molecules affects the behavior of the binding of 4-phenylimidazole to CYP158A2 and increases inhibition potency up to 2-fold compared to 4-phenylimidazole alone. We report here the crystal structure of the 4-phenylimidazole/malonate complex of CYP158A2 at 1.5 A. Two molecules of malonate used in crystallization are found above the single inhibitor molecule in the active site. Those two molecules are linked between the BC loop and beta 1-4/beta 6-1 strands via hydrogen bond interactions to stabilize the conformational changes of the BC loop and beta strands that take place upon inhibitor binding compared to the ligand-free structure we have reported previously. 4-Phenylimidazole can launch an extensive hydrogen-bonding network in the region of the F/G helices which may stabilize the conformational changes. Our findings clearly show that two molecules of malonate assist the inhibitor 4-phenylimidazole to assume a specific location producing more inhibition in the enzyme catalytic activity.

Topics: Binding Sites; Crystallization; Cytochrome P-450 Enzyme Inhibitors; Escherichia coli; Hydrogen Bonding; Imidazoles; Kinetics; Ligands; Malonates; Mixed Function Oxygenases; Models, Molecular; Naphthoquinones; Protein Conformation; Protein Structure, Secondary; Streptomyces coelicolor

A synthetic metabolic pathway has been constructed in vitro consisting of the type III polyketide synthase from Streptomyces coelicolor and peroxidases from soybean and Caldariomyces fumago (chloroperoxidase). This has resulted in the synthesis of the pentaketide flaviolin and its dimeric derivative, and a wide range of pyrones and their coupled derivatives with flaviolin, as well as their halogenated derivatives. The addition of acyl-CoA oxidase to the pathway prior to the polyketide synthase resulted in unsaturated pyrone side chains, further broadening the product spectrum that can be achieved. The approach developed in this work, therefore, provides a new model to exploit biocatalysis in the synthesis of complex natural product derivatives.

Topics: Acyl Coenzyme A; Acyltransferases; Malonyl Coenzyme A; Naphthoquinones; Pyrones; Streptomyces coelicolor

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Topics: Crystallization; Cytochrome P-450 Enzyme System; Dimerization; Naphthoquinones; Oxidation-Reduction; Protein Conformation; Streptomyces coelicolor

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.

Topics: Acyltransferases; Asparagine; Aspartic Acid; Binding Sites; Catalysis; Catalytic Domain; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Codon; Crystallography, X-Ray; Cysteine; Escherichia coli; Evolution, Molecular; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Naphthols; Naphthoquinones; Oxalic Acid; Polyketide Synthases; Protein Conformation; Protein Structure, Tertiary; Serine; Streptomyces; Streptomyces coelicolor

The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea "red variant" has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.

Topics: Bacterial Proteins; Cloning, Molecular; DNA Primers; Molecular Sequence Data; Molecular Structure; Multienzyme Complexes; Mutation; Naphthols; Naphthoquinones; Operon; Phenotype; Phylogeny; Pigments, Biological; Saccharopolyspora

Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.

Topics: Adult; Animals; Free Radicals; Humans; Macrophages, Peritoneal; Male; Melanins; Mice; Mice, Inbred BALB C; Monocytes; Mutation; Naphthols; Naphthoquinones; Respiratory Burst; Spores, Fungal; Sporothrix; Thiazoles; Ultraviolet Rays

The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.

Topics: Aspergillus oryzae; Cell-Free System; Colletotrichum; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; Malonyl Coenzyme A; Multienzyme Complexes; Naphthols; Naphthoquinones; Recombinant Proteins; Sodium Dodecyl Sulfate

Melanin biosynthesis in the human pathogen Wangiella dermatitidis was inhibited by tricyclazole, causing pentaketide melanin metabolites to accumulate in the cultures. One of these metabolites, scytalone, was racemic and thus different than the (+)-enantiomer from Verticillium dahliae. An albino mutant of W. dermatitidis metabolized scytalone to a pigment ultrastructurally identical to wild-type melanin. Cell-free homogenates of the wild type carried out typical reductive and dehydrative reactions with known melanin intermediates and the reductive reactions were inhibited by tricyclazole. Other reductive and dehydrative reactions that utilize flaviolin and 2-hydroxyjuglone were studied anaerobically with homogenates from both the wild type and the albino mutant. The homogenates converted flaviolin to 5-hydroxyscytalone and products identical to those obtained from 2-hydroxyjuglone. The albino, in culture, carried out the same reactions with 2-hydroxyjuglone but metabolized flaviolin to a number of unknown colored products apparently through oxidative reactions. Similarities between the melanin pathway and the flaviolin and 2-hydroxyjuglone branch pathways are discussed and tricyclazole is shown to inhibit reductive reactions with naphthols in the three pathways.

Topics: Melanins; Mitosporic Fungi; Naphthols; Naphthoquinones; Optical Rotation; Thiazoles

Melanin synthesis in the dematiaceous, polymorphic hyphomycete Wangiella dermatitidis, a human pathogen, was investigated by biochemical and physiological techniques. Mutants with a decrease or loss in melanin synthesis were induced and isolated. Melanin precursors were obtained from the mutants, purified, and then identified by comparison with authentic compounds from Verticillium dahliae . Isolation of scytalone , vermelone , flaviolin , and 1,8- dihydroxynaphthalene from the mutants of Wangiella dermatitidis, and cross-feeding of the mutants with those of Verticillium dahliae indicated that melanin synthesis in this organism took place by the pentaketide pathway. Melanin that formed in cell walls of an albino mutant treated with scytalone was identified in appearance to that in cell walls of the wild-type strain. This also suggested that pentaketide synthesis of melanin occurred in the fungus.

Topics: Melanins; Mitosporic Fungi; Mutation; Naphthols; Naphthoquinones; Species Specificity