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The aromatic polyketide tetracenomycin X (TcmX) was recently found to be a potent inhibitor of protein synthesis; its binding site is located in a unique locus within the tunnel of the large ribosomal subunit. The distinct mode of action makes this relatively narrow class of aromatic polyketides promising for drug development in the quest to prevent the spread of drug-resistant pathogens. Here we report the isolation and structure elucidation of a novel natural tetracenomycin X congener - 6-hydroxytetraceonomycin X (6-OH-TcmX). In contrast to TcmX, 6-OH-TcmX exhibited lower antimicrobial and cytotoxic activity, but comparable in vitro protein synthesis inhibition ability. A survey on spectral properties of tetracenomycins revealed profound differences in both UV-absorption and fluorescence spectra between TcmX and 6-OH-TcmX, suggesting a significant influence of 6-hydroxylation on the tetracenomycin X chromophore. Nonetheless, characteristic spectral properties of tetracenomycins make them suitable candidates for semi-synthetic drug development (e.g., for targeted delivery, chemical biology, or cell imaging)., (Copyright © 2021. Published by Elsevier B.V.)

Synthetic biology-based approaches have been employed to generate advanced natural product (NP) pathway intermediates to overcome obstacles in NP drug discovery and production. Type II polyketides (PK-IIs) comprise a major subclass of NPs that provide attractive structures for antimicrobial and anticancer drug development. Herein, we have assembled five biosynthetic pathways using a generalized operon design strategy in Streptomyces coelicolor M1152 to allow comparative analysis of metabolite production in an improved heterologous host. The work resulted in production of four distinct PK-II core structures, namely benzoisochromanequinone, angucycline, tetracenomycin, and pentangular compounds, which serve as precursors to diverse pharmaceutically important NPs. Our bottom-up design strategy provided evidence that the biosynthetic pathway of BE-7585A proceeds via an angucycline core structure, instead of rearrangement of an anthracycline aglycone, and led to the discovery of a novel 26-carbon pentangular polyketide. The synthetic biology platform presented here provides an opportunity for further controlled production of diverse PK-IIs in a heterologous host.

The secondary metabolite acarbose is used worldwide in the clinical treatment of diabetes mellitus type 2 patients. Acarbose is a - glucosidase inhibitor and supports patients to control their blood glucose as well as their serum insulin levels. The secondary metabolite is produced by strains of the class Actinobacteria, in particular from Actinoplanes sp. SE50/110, which is a progenitor of today`s production strains. Moreover, secondary metabolite clusters could also be identified in Streptomyces coelicoflavus ZG0656 as well as Streptomyces glaucescens GLA.O. In this study, the genome S. glaucescens GLA.O with focus on the acarbose biosynthesis cluster (gac-cluster) was analyzed. First, the tetracenomycin C and the 5`-hydroxy streptomycin gene clusters could be described completely. Then the gac gene region in S. glaucescens GLA.O is compared to the other known biosynthesis gene cluster. In comparison to Actinoplanes sp. SE50/110 the gac-cluster showed structural variances, like the missing homolog of the glycosyltransferase AcbD in the whole genome of S. glaucescens GLA.O. Due to the lack of the glycosyltransferase, it was of particular interest whether additional acarviose metabolites other than acarbose could be formed. For detection of acarviose metabolites biosynthesis the supernatant of S. glaucescens GLA.O grown in starch supplemented complex media was harvested at 72 and 96 hours. Although a homolog of the known glycosyltransferase is absent, the LC-MS-supported analysis revealed that a spectrum of acarviose metabolites was formed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Anthracyclines, such as doxorubicin, are effective anticancer drugs composed of a tetracyclic polyketide aglycone and one or more deoxysugar moieties, which play a critical role in their biological activity. A facile one-pot combinatorial biosynthetic system was developed for the generation of a range of glycosylated derivatives of anthracyclines. Cocultivation of Streptomyces venezuelae mutants producing two anthracycline aglycones with eight different nucleotide deoxysugar-producing S. venezuelae mutants that coexpress a substrate-flexible glycosyltransferase led to the generation of 16 aklavinone or ε-rhodomycinone glycosides containing diverse deoxysugar moieties, seven of which are new. This demonstrates the potential of the one-pot combinatorial biosynthetic system based on cocultivation as a facile biological tool capable of combining diverse aglycones and deoxysugars to generate structurally diverse polyketides carrying engineered sugars for drug discovery and development.

Phosphate metabolism regulates most of the life processes of microorganisms. In the present work we obtained and studied a Streptomyces lividans ppk/pstS double mutant, which lacks polyphosphate kinase (PPK) and the high-affinity phosphate-binding protein (PstS), impairing at the same time the intracellular storage of polyphosphate and the intake of new inorganic phosphate from a phosphate-limited medium, respectively. In some of the aspects analyzed, the ppk/pstS double mutant was more similar to the wt strain than was the single pstS mutant. The double mutant was thus able to grow in phosphate-limited media, whereas the pstS mutant required the addition of 1 mM phosphate under the assay conditions used. The double mutant was able to incorporate more than one fourth of the inorganic phosphate incorporated by the wt strain, whereas phosphate incorporation was almost completely impaired in the pstS mutant. Noteworthy, under phosphate limitation conditions, the double ppk/pstS mutant showed a higher production of the endogenous antibiotic actinorhodin and the heterologous antitumor 8-demethyl-tetracenomycin (up to 10-fold with respect to the wt strain), opening new possibilities for the use of this strain in the heterologous expression of antibiotic pathways., (© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel α-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the α-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-β-lactamase-type thioesterase (MβL-TE). The bifunctional MβL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon α-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of α-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MβL-TE is a dimanganese enzyme and requires both Mn(2+) cations for the observed activities. The MβL-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an α/β hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.

Streptomyces cinnamonensis C730.1 and C730.7, are industrially mutagenized strains that produce moderate and high levels of the polyketide polyether antibiotic monensin A, respectively, in an oil-based fermentation medium. The possibility that these strains could be used for high titer production of a heterologous polyketide product was investigated by expression of the entire tetracenomycin (TCM) biosynthetic pathway using an integrative plasmid, pSET154. Expression in C730.1 led to stable production of approximately 0.44 g/l TCM C (the final biosynthetic product) and approximately 2.69 g/l TCM A2 (the penultimate biosynthetic product), and resulted in a 40% decrease in monensin production. Expression in the C730.7 led to higher levels of TCMs, approximately 0.6 g/l TCM C and approximately 4.35 g/l TCM A2, without any detectable decrease in the higher titer monensin production. Abrogation of monensin production in this strain through deletion of the corresponding biosynthetic genes did not lead to higher levels of TCM products. In the case of the C730.7 host, 85% of the TCM C and virtually all of the TCM A2 were intracellular, suggesting feedback inhibition leads to the accumulation of the final pathway intermediate. These observations contrast those made for the native producer Streptomyces glaucescens where the predominant product is TCM C and TCM titers are significantly lower levels (approximately 0.3 g/l), and demonstrate the potential utility of S. cinnamonensis strains as heterologous hosts for high level expression of a variety of polyketide synthase derived products.

Effects of synthetic phenolic antioxidants (BHA, BHT, and TBHQ) on the methylene blue (MB) sensitized photooxidation of linoleic acid as compared with that of alpha-tocopherol have been studied. Their antioxidative mechanism was studied by both ESR spectroscopy in a 2,2,6,6-tetramethylpiperidone (TMPD)-methylene blue (MB) system and spectroscopic analysis of rubrene oxidation induced by a chemical source of singlet oxygen. Total singlet oxygen quenching rate constants (k(ox-Q)+k(q)) were determined using a steady state kinetic equation. TBHQ showed the strongest protective activity against the MB sensitized photooxidation of linoleic acid, followed by BHA and BHT. TBHQ (1 x 10(-3) M) exhibited 86.5% and 71.4% inhibition of peroxide and conjugated diene formations, respectively, in linoleic acid photooxidation after 60-min light illumination. The protective activity of TBHQ against the photosensitized oxidation of linoleic acid was almost comparable to that of alpha-tocopherol. The data obtained from ESR and rubrene oxidation studies clearly showed the strong singlet oxygen quenching ability of TBHQ. The k(ox-Q)+k(q) of BHA, BHT, and TBHQ were determined to be 3.37 x 10(7), 4.26 x 10(6), and 1.67 x 10(8) M(-1) s(-1), respectively. The k(ox-Q)+k(q) of TBHQ was within the same order of magnitude of that of alpha-tocopherol, a known efficient singlet oxygen quencher. There was a high negative correlation (r(2) = -0.991) between log (k(ox-Q)+k(q)) and reported oxidation potentials for the synthetic antioxidants, indicating their charge-transfer mechanism for singlet oxygen quenching. This is the 1st report on the kinetic study on k(ox-Q)+k(q) of TBHQ in methanol as compared with other commonly used commercial synthetic antioxidants and alpha-tocopherol.

Secondary alcohol metabolites and reactive oxygen species mediate cardiomyopathy induced by cumulative doses of antitumor anthracyclines, such as doxorubicin and epirubicin. Epirubicin exhibits a defective conversion to both toxic species, thereby inducing cardiotoxicity at doses higher than equiactive to doxorubicin; however, the gain in cardiac tolerability seems to be marginal compared with the magnitude of the metabolic defects of epirubicin. Cardiomyopathy may occur independent of toxic metabolites if a given anthracycline tends to accumulate in the heart; therefore, we characterized whether epirubicin showed an unusual accumulation in human myocardial strips incubated in plasma. Epirubicin exhibited a higher uptake and reached myocardial levels 2 times higher than those of doxorubicin. Epirubicin also showed a unique metabolization to doxorubicinolone, the product of epirubicin deglycosidation and carbonyl reduction. In diffusing from the strips to plasma, doxorubicinolone caused membrane permeation effects that augmented epirubicin elimination. Experiments with purified doxorubicinolone showed that the efflux of 1 mol doxorubicinolone promoted the concomitant elimination of as many as approximately 40 mol epirubicin. Doxorubicinolone could also diffuse from plasma back to the strips, causing a permeation effect that promoted epirubicin reuptake; however, this reverse process was slower and less potent. On balance, doxorubicinolone efflux diminished the epirubicin to doxorubicin accumulation ratio to approximately 1.5. These results suggest that the cardiac tolerability of epirubicin is limited by its accumulation in the heart and that such accumulation would be even higher in the absence of doxorubicinolone formation and efflux. These results may also serve guidelines for developing noncardiotoxic anthracyclines.

Benastatins are aromatic polyketides from Streptomyces spp. that efficiently inhibit glutathione-S-transferases and induce apoptosis. Their biosynthesis involves a type II polyketide synthase, and a ketoacyl synthase (KAS) III component (BenQ) similar to FabH that is crucial for providing and selecting the rare hexanoate PKS starter unit. The function of BenQ as a KAS III was experimentally proven by point mutation of the active site cysteine. In the mutant several novel short chain fatty acid derived penta- and hexacyclic benastatin derivatives with antiprolieferative activities are formed. Strategies for engineering benastatin biosynthesis were attempted. Synthetic starter units surrogates were not incorporated by block mutants, which suggests that the primer needs to be enzyme-bound. Thus, on the basis of KAS III crystal structures the three-dimensional structure of BenQ was modeled and the predicted substrate-binding tunnel was altered by individual mutations of potential gatekeeping residues (H95A and M99A). However, no significant changes in substrate specificity were observed, indicating that there are other or additional gatekeeping amino acid residues in BenQ or secondary factors including likely protein-protein interactions between BenQ and the PKS complex, and possible conformational changes in BenQ. Finally, a benQ null mutant was complemented with butyrate starter unit biosynthesis genes from the alnumycin biosynthesis gene cluster, which resulted in a great (10x) enhancement in the production of butyrate-primed hexacyclic benastatin derivatives. The successful generation of an alnumycin-benastatin FAS-PKS hybrid pathway highlights the potential of metabolic pathways, which may lead to novel potential therapeutics and increased yields of desired natural products.

Effects of phosphatidylcholine (PC) on the oxidation of oil by singlet oxygen in a W/O microemulsion and an emulsion food model containing tocopherol-stripped sunflower oil (TSSO) have been studied. The W/O microemulsion consisted of methylene chloride, butanol, and sodium dodecyl sulfate with PC (0, 250, 1000 ppm) and TSSO (0, 3.3, 16.5, 33 mg/mL). Production of singlet oxygen in the microemulsion was done chemically with hydrogen peroxide in the presence of sodium molybdate, and indirectly evaluated by rubrene oxidation at A529. The emulsion food model consisted of TSSO, distilled water, and xanthan gum with addition of 250 ppm PC and 4 ppm chlorophyll b, and was placed at 25 degrees C under fluorescent lights (1700 lux) for 24 h. The oxidation of TSSO was determined by thin-layer chromatography and values of conjugated dienoic acid (CDA) and peroxides (POV). PC significantly decreased the oxidation of rubrene and TSSO in the W/O microemulsion, but its content was decreased to approximately one-half by a 20-min reaction, indicating its degradation. This clearly shows that PC acted as an antioxidant via chemical quenching of singlet oxygen in the W/O microemulsion. A possible synergism between PC and TSSO was observed in singlet oxygen quenching in the microemulsion. PC also significantly decreased the chlorophyll-photosensitized oxidation of TSSO in the emulsion food model, possibly by singlet oxygen quenching. This study clearly suggested that PC be used as an antioxidant to improve the lipid oxidative stability of an emulsion food containing chlorophyll under light.

The benastatins, pradimicins, fredericamycins, and members of the griseorhodin/rubromycin family represent a structurally and functionally diverse group of long-chain polyphenols from actinomycetes. Comparison of their biosynthetic gene clusters (ben, prm, fdm, grh, rub) revealed that all loci harbor genes coding for a similar, yet uncharacterized, type of ketoreductases. In a phylogenetic survey of representative KRs involved in type II PKS systems, we found that it is generally possible to deduce the KR regiospecificity (C-9, C-15, C17) from the amino acid sequence and thus to predict the nature of the aromatic polyketide (e.g., angucycline, anthracycline, benzoisochromanequinones). We hypothezised that the new clade of KRs is characteristic for biosynthesis of polyphenols with an extended angular architecture we termed "pentangular". To test this hypothesis, we demonstrated the biogenetic relationship between benastatin and the structurally unrelated spiro ketal griseorhodin by generating a mutant producing collinone, a pentangular pathway intermediate. The benastatin pathway served as a model to characterize the KR. Gene inactivation of benL resulted in the formation of a series of 19-hydroxy benastatin and bequinostatin derivatives (e.g., benastatin K and benastatin L). These results clearly showed that BenL functions as a C-19 KR in pentangular pathways.

The entire gene locus encoding the biosynthesis of the potent glutathione-S-transferase inhibitors and apoptosis inducers benastatin A and B has been cloned and sequenced. The cluster identity was unequivocally proven by deletion of flanking regions and heterologous expression in S. albus and S. lividans. Inactivation and complementation experiments revealed that a KSIII component (BenQ) similar to FabH is crucial for providing and selecting the rare hexanoate PKS starter unit. In the absence of BenQ, several novel penta- and hexacyclic benastatin derivatives with antiproliferative activities are formed. In total, five new compounds were isolated and fully characterized, and the chemical analysis was confirmed by derivatization. The most intriguing observation is that the ben PKS can utilize typical straight and branched fatty acid synthase primers. If shorter straight-chain starters are utilized, the length of the polyketide backbone is increased, resulting in the formation of an extended, hexacyclic ring system reminiscent of proposed intermediates in the griseorhodin and fredericamycin pathways. Analysis and manipulation of the hybrid fatty acid polyketide pathway provides strong support for the hypothesis that the number of chain elongations is dependent on the total size of the polyketide chain that is accommodated in the PKS enzyme cavity. Our results also further substantiate the potential of metabolic engineering toward polyphenols with altered substituents and ring systems.

Aims: To investigate the diffusion and accumulation of doxorubicin metabolites in the ascites of patients with ovarian cancer following intravenous injection, as a model for intraperitoneal accumulation of drugs., Methods: The concentrations of doxorubicin and its metabolites [Doxorubicinol (Dox-ol), 7-deoxydoxorubicinolone (7d-Dox-ol-on) and 7-deoxydoxorubicinone (7d-Dox-on)] were measured using high-performance liquid chromatography in the serum and in the ascites of seven patients with recurrent ovarian carcinoma suffering from symptomatic ascites and treated with intravenous doxorubicin., Results: Doxorubicin metabolites accumulated in the peritoneal cavity. The concentrations of the doxorubicin metabolites were initially higher in the serum compared to the ascitic fluid, but following several hours the doxorubicin metabolites became higher in the ascites, and remained detectable in the ascites for up to 168h, long after disappearance from the serum., Conclusions: Doxorubicin metabolites accumulate in the ascites and are cleared more slowly from the peritoneal compartment than from the serum. Accumulation in the peritoneal cavity with prolonged half-life should be considered when administering medication in patients with ascites.

Combinatorial biosynthesis was applied to Streptomyces deoxysugar biosynthesis genes in order to reconstitute "unnatural natural gene clusters" for the biosynthesis of four D-deoxysugars (D-olivose, D-oliose, D-digitoxose, and D-boivinose). Expression of these gene clusters in Streptomyces albus 16F4 was used to prove the functionality of the designed clusters through the generation of glycosylated tetracenomycins. Three glycosylated tetracenomycins were generated and characterized, two of which (D-digitoxosyl-tetracenomycin C and D-boivinosyl-tetracenocmycin C) were novel compounds. The constructed gene clusters may be used to increase the capabilities of microorganisms to synthesize new deoxysugars and therefore to produce new glycosylated bioactive compounds.

Benzoic acid priming of the enterocin and actinorhodin type II polyketide synthase complexes was accomplished in vitro via an unprecedented type II nonribosomal peptide synthetase-like mechanism involving the benzoate:acyl carrier protein (ACP) ligase EncN and the ACP EncC. The transfer of the aryl acid to the ACP is ATP-dependent, yet coenzyme A-independent, as characterized with radiolabeled substrates and protein mass spectrometry. Subsequent transport of the ACP-bound aryl group to the native enterocin and the aberrant actinorhodin ketosynthase chain length factor heterodimers was further demonstrated, thereby demonstrating the potential of this biocatalyst for engineering diverse aryl-primed aromatic polyketide agents.

During biosynthesis of the anthracycline antitumor agents daunomycin, adriamycin, and aclacinomycin, the polyketide-derived tetracyclic aglycone is enzymatically glycosylated at the C7-OH by dedicated glycosyltransferases (Gtfs) that transfer L-2,3,6-trideoxy-3-aminohexoses. In aclacinomycins, the first deoxyhexose is predicted to be transferred via AknS action, then subjected to further elongation to a trisaccharide by the subsequent Gtf, AknK. We report here that purified AknS has very low activity in the absence of the adjacently encoded AknT; however, at a 3:1 ratio, AknT stimulates AknS k(cat) by 40-fold up to 0.22 min(-1) for transfer of L-2-deoxyfucose (2-dF) to the aglycone aklavinone. It is likely that several other Gtfs that glycosylate polyketide aglycones also act as two-component catalytic systems. Incubations of purified AknS/AknT/AknK with two aglycones and two dTDP-2-deoxyhexoses produced previously uncharacterized anthracycline disaccharides.

Sugar biosynthesis cassette genes have been used to construct plasmids directing the biosynthesis of branched-chain deoxysugars: pFL942 (NDP-L-mycarose), pFL947 (NDP-4-deacetyl-L-chromose B), and pFL946/pFL954 (NDP-2,3,4-tridemethyl-L-nogalose). Expression of pFL942 and pFL947 in S. lividans 16F4, which harbors genes for elloramycinone biosynthesis and the flexible ElmGT glycosyltransferase of the elloramycin biosynthetic pathway, led to the formation of two compounds: 8-alpha-L-mycarosyl-elloramycinone and 8-demethyl-8-(4-deacetyl)-alpha-L-chromosyl-tetracenomycin C, respectively. Expression of pFL946 or pFL954 failed to produce detectable amounts of a novel glycosylated tetracenomycin derivative. Formation of these two compounds represents examples of the sugar cosubstrate flexibility of the ElmGT glycosyltransferase. The use of these cassette plasmids also provided insights into the substrate flexibility of deoxysugar biosynthesis enzymes as the C-methyltransferases EryBIII and MtmC, the epimerases OleL and EryBVII, and the 4-ketoreductases EryBIV and OleU.

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.

Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens. Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH. In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis. We have shown that sgFabH activity is present in S. glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo. Isotope incorporation studies with [CD3]acetate and [13CD3]acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C. These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process. Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S. glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E. coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S. glaucescens PKS ACP). In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM). This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes. Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols. The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes. In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.

The rdm genes B, C and E from Streptomyces purpurascens encode enzymes that tailor aklavinone and aclacinomycins. We report that in addition to hydroxylation of aklavinone to epsilon-rhodomycinone, RdmE (aklavinone-11-hydroxylase) hydroxylated 11-deoxy-beta-rhodomycinone to beta-rhodomycinone both in vivo and in vitro. 15-Demethoxyaklavinone and decarbomethoxyaklavinone did not serve as substrates. RdmC (aclacinomycin methyl esterase) converted aclacinomycin T (AcmT) to 15-demethoxyaclacinomycin T, which was in turn converted to 10-decarbomethoxyaclacinomycin T and then to rhodomycin B by RdmB (aclacinomycin-10-hydroxylase). RdmC and RdmB were most active on AcmT, the one-sugar derivative, with their activity decreasing by 70-90% on two- and three-sugar aclacinomycins. Aclacinomycin A competitively inhibited the AcmT modifications at C-10. The results presented here suggest that in vivo the modifications at C-10 take place principally after addition of the first sugar.

Background: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC)., Results: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC., Conclusions: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.

Bacterial strains grown in the presence of low concentrations of dimethyl sulfoxide (DMSO) exhibit significant qualitative and quantitative alterations in the production of secondary metabolites. This effect was confirmed for a variety of biosynthetic families, including chloramphenicol (chorismate), thiostrepton (peptide) and tetracenomycin (polyketide), and for natural and recombinant strains of streptomycetes; a similar effect was seen with antibiotic-producing bacilli such as B. circulans. Increase in antibiotic production was not the result of a change in the growth rate of these organisms, since yields of biomass were similar in media with and without DMSO (up to 3%). We suggest that the addition of compounds such as DMSO provides a means of examining the full biosynthetic potential of microbes and might be used to promote secondary metabolite production. The mode of action of DMSO is not known, but in the cases studied it may act at the level of translation.

Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8-demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product.

Drug-transporting P-glycoproteins are abundantly present in the liver and the intestinal wall. We have now investigated their role in the biliary and intestinal secretion of the anticancer drugs doxorubicin (unlabeled: 5 mg/kg) and vinblastine ((3)H-labeled: 1 mg/kg) i.v. administered to wild-type and mdr1a P-glycoprotein knockout [mdr1a(-/-)] mice. At 90 min after drug administration, levels of unchanged drug and metabolites in plasma, intestinal contents, and bile were determined by high-performance liquid chromatography and radioactivity by liquid scintillation counting. The bile of both wild-type and mdr1a(-/-) mice contained only minor amounts of unchanged vinblastine, whereas the total biliary secretion of unknown (3)H-labeled breakdown products was about 25 to 30% of the dose. The direct secretion of unchanged vinblastine through the gut wall was 6.7 and 3.3% of the dose in wild-type and mdr1a(-/-) mice, respectively. The biliary secretion of unchanged doxorubicin decreased from 13.3% of the dose to only 2.4% in the absence of mdr1a P-glycoprotein. Approximately 10% of the dose was secreted as unchanged doxorubicin into the intestinal contents of both types of mice. Thus, the absence of mdr1a P-glycoprotein affects the fate of vinblastine chiefly by diminishing secretion into the lumen of the small intestine, whereas it affects the fate of doxorubicin chiefly by diminishing secretion of parent drug into bile.

The knowledge about molecular factors driving simple ligand-DNA interactions is still limited. The aim of the present study was to investigate the electrostatic and non-electrostatic contributions to the binding free energies of anthracycline compounds with DNA. Theoretical calculations based on continuum methods (Poisson-Boltzmann and solvent accessible surface area) were performed to estimate the binding free energies of five selected anthracycline ligands (daunomycin, adriamycin, 9-deoxyadriamycin, hydroxyrubicin, and adriamycinone) to DNA. The free energy calculations also took into account the conformational change that DNA undergoes upon ligand binding. This conformational change appeared to be very important for estimating absolute free energies of binding. Our studies revealed that the absolute values of all computed contributions to the binding free energy were quite large compared to the total free energy of binding. However, the sum of these large positive and negative values produced a small negative value of the free energy around -10 kcal/mol. This value is in good agreement with experimental data. Experimental values for relative binding free energies were also reproduced for charged ligands by our calculations. Together, it was found that the driving force for ligand-DNA complex formation is the non-polar interaction between the ligand and DNA even if the ligand is positively charged., (Copyright 1997 Academic Press Limited.)

The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.

The role of enzymes in the reductive activation of various chemotherapeutic agents is an area of considerable interest in studies to better understand the selective toxicities of these agents. Xanthine dehydrogenase (XDH) is an enzyme capable of reductive activation of chemotherapeutic agents. Previously, this enzyme has not been extensively studied because of difficulties in its isolation. We recently isolated this enzyme from EMT6 mouse mammary carcinoma cells and showed that this enzyme is capable of activating mitomycin C. In this study, we examined whether XDH could activate the clinically important antineoplastic agent, doxorubicin. Drug activation was determined under aerobic and hypoxic conditions and at various pHs in order to simulate the different environments found in solid tumors. The results of these studies show that XDH reacts with doxorubicin via a two-electron reduction. This reduction is different from the modified and more extensively studied form of the enzyme, xanthine oxidase (XO), which reacts with doxorubicin via a one-electron reduction. Under hypoxic conditions, the formation of large quantities of 7-deoxydoxorubicin aglycone, a deactivation product of doxorubicin metabolism, may serve to moderate doxorubicin's antineoplastic activity. Under aerobic conditions, however, XDH activation led to a greater rate of formation of oxygen radicals than XO thereby possibly potentiating doxorubicin's cytotoxicity to aerobic tumor cells. Kinetic constants were determined for doxorubicin activation by XDH.

The molecular basis of the adriamycin (AQ)-dependent development of cardiotoxicity is still far from being clear. In contrast to our incomplete understanding of the organ-specific mechanism mitochondria are unequivocally accepted as the locus where the molecular disorder is triggered. A growing number of reports intimate the establishment of unbalanced oxygen activation through heart mitochondria in the presence of anthraquinones. In fact, in contrast to liver mitochondria, isolated heart mitochondria have been unequivocally shown to shuttle single electrons to AQ, giving rise to O2.- formation by autoxidizing AQ. semiquinones. Earlier we have demonstrated the involvement of the exogenous NADH dehydrogenase in this deleterious electron deviation from the respiratory chain. This enzyme that is associated with complex I of the respiratory chain catalyzes the oxidation of cytosolic NADH. AQ activation through isolated heart mitochondria was reported to require the external addition of NADH, suggesting a flux of reducing equivalents from NADH to AQ in the cytosol. Unlike heart mitochondria, intact liver mitochondria, which are lacking this NADH-related pathway of reducing equivalents from the cytosol to the respiratory chain, cannot be made to activate AQ to semiquinones by NADH or any other substrate of respiration. It appears, therefore, that the exogenous NADH dehydrogenase of heart mitochondria exerts a key function in the myocardial toxicogenesis of anthraquinones via oxygen activation through semireduced AQ. Assessing the toxicological significance of the exogenous NADH dehydrogenase in AQ-related heart injury requires analysis of reaction products and their impact on vital bioenergetic functions, such as energy gain from the oxidation of respiratory substrates. We have applied ESR technique to analyze the identity and possible interactions of radical species emerging from NADH-respiring heart mitochondria in the presence of AQ. The following metabolic steps occur causing depression of energy metabolism in the cardiac tissue. After one-electron transfer to the parent hydrophilic anthraquinone molecule destabilization of the radical formed causes cleavage of the sugar residue. Accumulation of the lipophilic aglycone metabolite in the inner mitochondrial membrane diverts electrons from the regular pathway to electron acceptors out of sequence such as H2O2. HO. radicals are formed and affect the functional integrity of energy-linked respiration. The key and possibly initiating role of the exogenous NADH dehydrogenase of cardiac mitochondria in this reaction pathway provides a rationale to explain the selective cardiotoxic potency of the cytostatic anthraquinone glycosides.

TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four-ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S-adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6-methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates.

The production of semiquinone free radicals has been measured by electron paramagnetic resonance spectroscopy (EPR) in Chinese hamster ovary cells in which 7-hydroxy daunorubicin aglycone had been incorporated. The highly lipophilic daunorubicin aglycone was incorporated into the cellular membrane by swirling a cell suspension over a thin layer of daunorubicin aglycone. Thus, the observed semiquinone free radical was likely formed directly in the lipophilic environment of the cellular membrane. The linewidth of the observed EPR signal suggested that a neutral protonated semiquinone species was formed. In the presence of the cell-impermeant paramagnetic line broadening agent chromium(III) oxalate, no detectable signal was observed. This result indicates that even though the semiquinone is embedded in the membrane, it is still partly accessible to the external chromium(III) oxalate. Analysis of chloroform extracts of the cells after EPR experiments indicated that daunorubicin aglycone was extensively metabolized. The results of a growth inhibition assay carried out on cells into which daunorubicin aglycone had been incorporated showed almost no effect on cell growth. This result indicates that in spite of significant daunorubicin aglycone-induced radical formation taking place directly in the cell membrane, little cell damage results.

A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae Tü2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C.

Nucleotide sequence analysis of the tcmG gene has suggested that the TcmG protein is responsible for the triple-hydroxylation of tetracenomycin (Tcm) A2 to Tcm C in Streptomyces glaucescens (Decker, H., Motamedi, H., and Hutchinson, C.R. (1993) J. Bacteriol. 175, 3876-3886). The heterologous expression of the tcmG gene in Streptomyces lividans and the purification and characterization of TcmG protein, which we have named Tcm A2 oxygenase, are described here. NH2-terminal amino acid analysis of the purified enzyme led to the revision of the translational start site of tcmG to a TTG, 33 base pairs downstream of the GTG site assigned initially on the basis of nucleotide sequence analysis. Tcm A2 oxygenase is a monomeric protein in solution and contains 1 mol of non-covalently bound FAD; the apoenzyme can be partially reconstituted in vitro by addition of FAD. Tcm A2 oxygenase exhibits an optimal pH of 9.0-9.5 and prefers NADPH over NADH as an electron donor. The apparent K'm of the enzyme for Tcm A2, NADH, and NADPH are 1.81 +/- 0.38, 260 +/- 19, and 82.1 +/- 17 microM, respectively, and the apparent V'max for the reaction is 14.7 +/- 1.1 nmol Tcm C/min.mg. Purification and characterization of Tcm A2 oxygenase provide direct evidence to support the notion that the angular hydroxy groups of naphthacenequinones like Tcm C are introduced from 18O2 via a mono- or dioxygenase process.

Adriamycin and related anthracyclines are potent oncolytic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone metabolites of Adriamycin (5-20 microM) induce a Ca(2+)-dependent increase in the permeability of the inner mitochondrial membrane of both heart and liver mitochondria to small (< 1,500 Da) solutes; this phenomenon is accompanied by release of mitochondrial Ca2+, mitochondrial swelling, collapse of the membrane potential, oxidation of mitochondrial pyridine nucleotides [NAD(P)H], uncoupling, and a transition from the condensed to the orthodox conformation and is inhibited by ATP, dithiothreitol, the immunosuppressant cyclosporin A, and the ubiquitous polyamine spermine. Aglycones also modify mitochondrial sulfhydryl groups and induce a Ca2+ independent oxidation of mitochondrial NAD(P)H which appears to reflect electron transport from NADH to oxygen, mediated by the aglycones and resulting in the production of superoxide (O2-). Selenium deficiency and butylated hydroxytoluene inhibit aglycone-induced Ca2+ release from liver, but not heart, mitochondria, suggesting that the interactions of the aglycones with mitochondria differ in these two tissues. It can be proposed that the effects of Adriamycin aglycones on heart mitochondria are responsible for the cardiotoxicity of the parent drug.

The gene encoding aklavinone 11-hydroxylase of Streptomyces peucetius subsp. caesius ATCC 27952 was cloned and sequenced. The deduced amino acid sequence of the gene contains at least two common motifs of well-conserved amino acid sequences of several flavin-type bacterial hydroxylases. The hydroxylase gene is apparently transcribed from a single transcriptional start point. The phenotype of a dnrF mutant generated by gene disruption supports the idea that the dnrF gene encodes aklavinone 11-hydroxylase.

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.

A DNA segment cloned from Streptomyces purpurascens ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase caused S. galilaeus ATCC 31615 to produce new anthracyclines. When transformed with certain sub-clones of this segment, the host produced glycosides of epsilon-rhodomycinone, beta-rhodomycinone, 10-demethoxycarbonylaklavinone and 11-deoxy-beta-rhodomycinone in addition to those of aklavinone, the natural anthracyclines of S. galilaeus. The first two compounds are S. purpurascens products and the other two are novel compounds that conceptually are structural hybrids between S. galilaeus and S. purpurascens products. Three glycosides of one of the novel aglycones, 11-deoxy-beta-rhodomycinone, were purified and found to possess cytotoxic activity against L1210 mouse leukaemia cells. Separate regions of the cloned S. purpurascens DNA are responsible for modification of the S. galilaeus host product at the 10- and 11-positions.

A 3.4-kb BamHI fragment that is assumed to be a part of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus 3AR-33 and contains the genes required for the early stage of polyketide biosynthesis was sequenced. The nucleotide sequence of the region that hybridizes to the actIII probe reveals the presence of a gene, aknA, whose deduced protein product is very similar to the ActIII protein and other known oxidoreductases. The predicted AknA protein is believed to be responsible for catalyzing the reduction of the keto group at the ninth carbon from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The predicted AknA protein has a calculated molecular mass of 27,197 Da (261 amino acids) and the highly conserved sequence Gly-Xaa-Gly-Xaa-Xaa-Ala commonly seen in oxidoreductases. Cloning and sequence analysis of the aknA region of the 2-hydroxyaklavinone-producing strain S. galilaeus ANR-58 identified an alteration in the gene, confirming that the aknA gene is essential for aklavinone biosynthesis.

The development of microorganisms with improved antibiotic production is an important goal in the commercialization of new pharmaceuticals or in lowering the cost of established drugs. We report a way to achieve this for biosynthetic intermediates of an antibiotic made by the polyketide pathway whose earliest steps involve a Type II multienzyme complex. Introduction of the tcmKLM beta-ketoacyl: ACP synthase and acyl carrier protein (ACP) genes or just the tcmM ACP gene into the tetracenomycin (Tcm) C-producing Streptomyces glaucescens wild-type strain, or its tcmN or tcmO blocked mutants, on high copy vectors under the control of strong promoters caused a 2 to 30-fold overproduction of Tcm D3 and some other biosynthetic intermediates (or shunt products) and a 25 to 30% increase in Tcm C production relative to the control strains carrying the plasmid vector only. However, Tcm C production was not greater than that obtained with the vector-free wild-type strain. The unexpected effect of increased ACP on Tcm D3 production suggests that the level of this protein can influence either the activity or level of the three other components of the Tcm polyketide synthase.

Certain mutations in the tcmVI region of the Streptomyces glaucescens chromosome affect formation of the D ring of the polyketide antibiotic tetracenomycin C (TCM C). This region lies immediately upstream from the TCM C polyketide synthase genes (tcmKLM), and the nucleotide sequence reveals the presence of three small genes, tcmH, tcmI, and tcmJ. On the basis of the phenotypes of mutants and the effects of these genes, when coupled on a plasmid with the tcmKLMN177 genes (tcmN177 is a 3'-truncated version of tcmN), on the production of TCM intermediates in a TCM- mutant, the tcmH gene encodes the C-5 monooxygenase that converts TCM F1 to TCM D3, the tcmI gene encodes the D-ring cyclase that converts TCM F2 to TCM F1 (mutations in this gene are responsible for the type VI phenotype), and the tcmJ gene most likely encodes the B-ring cyclase that acts in the biosynthesis of TCM F2. Furthermore, it appears that the N-terminal domain of the tcmN gene product (encoded by the tcmN177 gene) acts later in the biosynthesis of TCM F2 than the product of tcmJ, suggesting that the N-terminal domain of the TcmN protein is the C-ring cyclase.

Tetracenomycin (Tcm) F2 cyclase, which catalyzes the cyclization of the anthrone Tcm F2 to the naphthacenone Tcm F1 in the biosynthesis of the anthracycline antibiotic Tcm C in Streptomyces glaucescens, has been purified to homogeneity and characterized. The N-terminal sequence of the enzyme establishes that it is encoded by the tcmI gene, whose deduced product has a molecular weight of 12,728. SDS-PAGE analysis gave a single band with a molecular weight of 12,500, whereas gel-filtration chromatography yielded a molecular weight of 37,500, indicating that the Tcm F2 cyclase is a homotrimer in solution. Under pH > or = 8.0, the enzyme catalyzes the cyclization of Tcm F2 to Tcm F1 and has a Km of 121 +/- 18.2 microM and Vmax of 704 +/- 62.3 nmol.min-1.mg-1. In contrast, under pH < or = 6.5, it catalyzes the cyclization of Tcm F2 to 9-decarboxy Tcm F1, a known shunt metabolite of the Tcm C biosynthetic pathway. Tcm F2 cyclase represents the first discrete enzyme for carbon-carbon bond formation via an intramolecular aldol condensation-dehydration mechanism, a key biochemical operation proposed in the early steps of the biosynthesis of all aromatic polyketides.

Unusually high electron transfer rates in Aspergillus niger glucose oxidase catalyzed oxidation of glucose using 5,6:11,12-Bis(dithio)tetracene (TTT), 1,2-dimethyltetraselenafulvalene (DMTSF) and tetrathiafulvalene (TTF) were observed. At pH 7.0 oxidation rate constants (TN/Km) in the range from 1.0.10(7) to 8.7.10(7) M.s-1 were deduced from experimental data. One of the investigated mediators, DMTSF, has been used for electrocatalytical glucose oxidation on graphite at a potential of 0.3 V vs. a standard calomel electrode (SCE). The prepared bioelectrodes have a sensitivity of 1.3 microA/(cm2.mM), a pH optimum at 6.5-7.0, and a linear range which covers the relevant range for monitoring physiological levels of glucose. The bioelectrodes are stable for more than one month.

This report describes the fermentation, isolation, and structural elucidation of tetracenomycin (Tcm) F2 [2], a metabolite produced by a blocked mutant strain WMH1092 of the Tcm C [1] producer Streptomyces glaucescens and by the recombinant strain S. glaucescens WMH1077 (pWHM722). Elucidation of the Tcm F2 structure shows that 2 is the earliest intermediate identified to date in the biosynthesis of 1. This is supported by the fact that 2 is very efficiently biotransformed to 1 by the S. glaucescens WMH1068 strain and is enzymatically converted to Tcm F1 [3] and to Tcm D3 [4], a known intermediate of Tcm C biosynthesis.

Tetracenomycin (Tcm) F1 monooxygenase, which catalyzes the oxidation of the naphthacenone Tcm F1 to the 5,12-naphthacenequinone Tcm D3 in the biosynthesis of the anthracycline antibiotic Tcm C in Streptomyces glaucescens, has been purified to homogeneity and characterized. Gel filtration chromatography yields a molecular weight of 37,500 whereas SDS-PAGE gives a single band with a molecular weight of 12,500, indicating that the Tcm F1 monooxygenase is a homotrimer in solution. The N-terminal sequence of the enzyme establishes that it is encoded by the tcmH gene. The monooxygenase displays an optimal pH of 7.5 and has a Km of 7.47 +/- 0.67 microM and Vmax of 473 +/- 10 nmol.min-1.mg-1. Formally, the Tcm F1 monooxygenase can be classified as an internal monooxygenase that requires only O2 for the enzymatic oxidation. Yet, it apparently does not possess any of the prosthetic groups of known monooxygenases, such as flavin or heme groups, nor does it utilize metal ions. It is inactivated by p-chloromercuribenzoic acid, N-ethylmaleimide, and diethyl pyrocarbonate, suggesting that sulfhydryl groups and histidine residues are essential for the enzyme activity.