Antimicrobial tunicamycin derivatives from the deep sea-derived Streptomyces xinghaiensis SCSIO S15077.

Tunicamycin E (1), featuring a methyl substitution at C-10', was isolated from marine-derived Streptomyces xinghaiensis SCSIO S15077 originated from the South China Sea sediment together with six known compounds, tunicamycin B (2), tunicamycin X (3), tunicamycin A (4), streptovirudin D2 (5), tunicamycin C (6), and tunicamycin C3 (7). The structure of compound 1 was elucidated by detailed spectroscopic data analyses. All the compounds exhibited strong to moderate antibacterial activity against Gram-positive bacteria Bacillus thuringiensis BT01 and B. thuringiensis W102 with MIC values ranging from 0.008 to 2 µg/mL. Moreover, compounds 1-7 exhibited moderate antifungal activity against Candida albicans ATCC 96901 and C. albicans CMCC (F) 98001 with MIC values ranging from 2 to 32 µg/mL. This is the first report that tunicamycins exhibit antimicrobial activities against B. thuringiensis, C. albicans CMCC (F) 98001 and a fluconazole resistant strain C. albicans ATCC 96901.

Quinovosamycins: new tunicamycin-type antibiotics in which the α, ß-1″,11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine.

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall polysaccharides. The genetic requirement for TUN production is well-established. Using two genes unique to the TUN pathway (tunB and tunD) as probes we identified four new prospective TUN-producing strains. Chemical analysis showed that one strain, Streptomyces niger NRRL B-3857, produces TUN plus new compounds, named quinovosamycins (QVMs). QVMs are structurally akin to TUN, but uniquely in the 1″,11'-HexNAc sugar head group, which is invariably d-GlcNAc for the known TUN, but is d-QuiNAc for the QVM. Surprisingly, this modification has only a minor effect on either the inhibitory or antimicrobial properties of QVM and TUN. These findings have unexpected consequences for TUN/QVM biosynthesis, and for the specificity of the PPHP enzyme family.

Several intermediate forms in the processing of rat lutropin subunits as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

To study the biosynthesis in situ of lutropin (LH) subunits, anterior pituitary cells in culture were employed. The cells were incubated in the presence of [35S]methionine. Labelled polypeptides, immunologically related to alpha and LH beta subunits, were isolated by specific immunoprecipitation, then analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and revealed by fluorography. Electrophoretic analysis of immunoprecipitates is a crucial step in this methodology. It permits simultaneous purification, characterization and accurate quantitation of the radioactivity specifically incorporated into LH subunits. Also, using SDS-PAGE, it is possible to isolate and identify the different processed forms of LH subunits.

Relationship of the structure and biological activity of the natural homologues of tunicamycin.

The antibiotic tunicamycin was separated into 16 different components using reversed-phase high performance liquid chromatography. The effect of the eight major tunicamycin homologues on protein glycosylation and protein biosynthesis was examined. All homologues tested inhibited lipid-mediated protein glycosylation in chick or mouse fibroblasts. These homologues also inhibited the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichyl phosphate in chick liver microsome preparations, whereas the transfer of mannose from GDP-mannose to the lipid acceptor was hardly affected. The inhibition of protein glycosylation in fibroblasts or in microsomal preparations was concentration-dependent and maximum inhibition occurred at different concentrations for different homologues. The eight homologues differed in their ability to cause inhibition of protein synthesis.

Biological activities of isolated tunicamycin and streptovirudin fractions.

The nucleoside antibiotics tunicamycin and streptovirudin were separated by high-performance liquid chromatography into a series of 256-nm-absorbing peaks. Most of the streptovirudin peaks eluted from a Biosil ODS column earlier than those of tunicamycin, indicating that they were less hydrophobic. With the exception of the first peak, 17 other tunicamycin peaks were potent inhibitors of the formation of dolichylpyrophosphoryl-N-acetylglucosamine with 50% inhibition of the solubilized GlcNAc-1-P transferase requiring about 10 ng of antibiotic per mL. These fractions also inhibited the synthesis of dolichylphosphorylglucose, but in these cases about 500 ng/mL was necessary to achieve 50% inhibition. In MDCK cells in culture, the four major tunicamycin peaks inhibited the incorporation of [2-(3)H]mannose into protein by 50% at about 0.2-0.5 microgram/mL, but [3H]leucine incorporation into protein was unaffected, except at high levels of antibiotic (5-10 microgram/mL). Essentially the same results were observed with the streptovirudin fractions except that they were somewhat less active and some inhibition of protein synthesis was observed with several of these peaks.

Biological activities of the two major components of tunicamycin.

Tunicamycin, an antibiotic that inhibits the transfer of N-acetyglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol monophosphate and thereby blocks the formation of protein-carbohydrate linkages of the N-glycosidic type, is not a single compound but a mixture of homologous antibiotics. Two major and eight minor homologs have been identified, all of which possess the ability to inhibit protein glycosylation. The biological activities of the two major components of tunicamycin were investigated and found to differ in their ability to inhibit protein glycosylation and in their effectiveness to inhibit protein synthesis. When completely blocking mannose incorporation into protein, one homolog inhibited protein synthesis by 50% while the other had only a negligible effect. The results demonstrate that differences in biological activity can be discriminated among tunicamycin homologs.

Holomycin and an antibiotic (MM 19290) related to tunicamycin, metabolites of Streptomyces clavuligerus.

Streptomyces clavuligerus produces penicillin N, several cephalosporins and the beta-lactamase inhibitor clavulanic acid. The detection, isolation and properties of further metabolites of this culture, MM 21801 and MM 19290, are described. MM 21801 was identified as the antibiotic holomycin. MM 19290 was shown to be related to tunicamycin, an antibiotic complex obtained from cultures of Streptomyces lysosuperificus.