Biosynthesis of Diverse Type II Polyketide Core Structures in Streptomyces coelicolor M1152.

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.

(1-4)-Thiodisaccharides as anticancer agents. Part 5. Evaluation of anticancer activity and investigation of mechanism of action.

(1-4)-Thiodisaccharides, thiosugars with the 1-4-thio bridge, were recently shown to induce oxidative stress, as well as, apoptosis in cancer cells in the low micromolar range; however, the detailed mechanism of their anticancer action still remains unknown. In order to clarify the mechanism of (1-4)- thiodisaccharides action, we performed a series of tests including cytotoxic, clonogenic and apoptosis assays using an in vitro glioma cancer model with one ATCC cell line U87 and two novel glioma cell lines derived from cancer patients - H6PX and H7PX. We also evaluated the ability of (1-4)-thiodisaccharides to interfere with protein folding and synthesis processes, as well as, the thioredoxin system. (1-4)-thiodisaccharides induced glioma cell death, which were found to be accompanied with endoplasmic reticulum stress, inhibition of global protein synthesis, reduced overall cellular thiol level and thioredoxin reductase activity. We also performed a RT-PCR and Elisa analysis of (1-4)-thiodisaccharides-treated glioma cells to identify any changes within the pathway affected by (1-4)-thiodisaccharides. We observed a significant increase of expression in key markers of endoplasmic reticulum stress and pro-apoptotic protein, FASLG. We proposed that (1-4)-thiodisaccharides react with cellular thiols and disturb any cellular thiol-depended processes like thioredoxin system or protein folding.

Synthesis and antimicrobial activity of 6-sulfo-6-deoxy-D-glucosamine and its derivatives.

6-Sulfo-6-deoxy-D-glucosamine (GlcN6S), 6-sulfo-6-deoxy-D-glucosaminitol (ADGS) and their N-acetyl and methyl ester derivatives have been synthesized and tested as inhibitors of enzymes catalyzing reactions of the UDP-GlcNAc pathway in bacteria and yeasts. GlcN6S and ADGS at micromolar concentrations inhibited glucosamine-6-phosphate (GlcN6P) synthase of microbial origin. The former was also inhibitory towards fungal GlcN6P N-acetyl transferase, but at millimolar concentrations. Both compounds and their N-acetyl derivatives exhibited antimicrobial in vitro activity, with MICs in the 0.125-2.0 mg mL-1 range. Antibacterial but not antifungal activity of GlcN6S was potentiated by D-glucosamine and a synergistic antibacterial effect was observed for combination of ADGP and a dipeptide Nva-FMDP.

Insights into Complex Oxidation during BE-7585A Biosynthesis: Structural Determination and Analysis of the Polyketide Monooxygenase BexE.

Cores of aromatic polyketides are essential for their biological activities. Most type II polyketide synthases (PKSs) biosynthesize these core structures involving the minimal PKS, a PKS-associated ketoreductase (KR) and aromatases/cyclases (ARO/CYCs). Oxygenases (OXYs) are rarely involved. BE-7585A is an anticancer polyketide with an angucyclic core. (13)C isotope labeling experiments suggest that its angucyclic core may arise from an oxidative rearrangement of a linear anthracyclinone. Here, we present the crystal structure and functional analysis of BexE, the oxygenase proposed to catalyze this key oxidative rearrangement step that generates the angucyclinone framework. Biochemical assays using various linear anthracyclinone model compounds combined with docking simulations narrowed down the substrate of BexE to be an immediate precursor of aklaviketone, possibly 12-deoxy-aklaviketone. The structural analysis, docking simulations, and biochemical assays provide insights into the role of BexE in BE-7585A biosynthesis and lay the groundwork for engineering such framework-modifying enzymes in type II PKSs.

[Thiosugars used as drugs]. / Tiocukry jako leki.

Thiosugars are carbohydrate analogs in which one or few of the oxygen atoms were replaced by sulfur. The sulfur atom which is present in the furan and pyran structures, changes biological properties of carbohydrates, as compared to their oxygen analogs. Among others, thiosugars are effective inhibitors of various cellular and enzymatic pathways and also have great therapeutic potential. They are used as a drugs in diabetes and infectious diseases treatment. Recent evidence suggests that these compounds may have therapeutic properties and be also used in the treatment of some pathological conditions, including cancer diseases. This research are aimed towards the development and improvement of the current methods of synthesis of new thiosugars through stabilization of sulfur bonds and in vitro and in vivo analysis of their potential therapeutic properties. In this work the summary of the latest reports about thiosugars and their application in the medicine is presented for the first time in the Polish language literature.

Binding interaction of SGLT with sugar and thiosugar by the molecular dynamics simulation.

The human sodium-glucose co-transporter 2 (hSGLT2) is a transporter responsible for reabsorption of glucose in the proximal convoluted tubule of the kidney. hSGLT2 inhibitors, including luseogliflozin, have been developed as drugs for type 2 diabetes mellitus. Only luseogliflozin contains a thiosugar ring in its chemical structure, while other hSGLT2 inhibitors contain glucose rings. Consequently, we focused on the binding interactions of hSGLT2 with sugars and thiosugars. We first revealed that the binding affinities of thiosugars are stronger than those of sugars through molecular dynamics simulations of Vibrio parahaemolyticus, sodium-galactose co-transporter, and human hSGLT2. We then demonstrated that Na(+) dissociates from the protein to the cytoplasmic solution more slowly in the thiosugar system than in the sugar system. These differences between sugars and thiosugars are discussed on the basis of the different binding modes due to the atom at the 5-position of the sugar and thiosugar rings. Finally, as a result of Na(+) dissociation, we suggest that the dissociation of thiosugars is slower than that of sugars.

Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis.

Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon-sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery of primary metabolism for the biosynthesis of sulphur-containing natural products is probably a general strategy found in nature.

A biosynthetic pathway for BE-7585A, a 2-thiosugar-containing angucycline-type natural product.

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using (13)C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning approximately 30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in Escherichia coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phosphate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.