A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Identification of Late-Stage Glycosylation Steps in the Biosynthetic Pathway of the Anthracycline Nogalamycin
Tekijät: Siitonen V, Claesson M, Patrikainen P, Aromaa M, Mantsala P, Schneider G, Metsa-Ketela M
Kustantaja: WILEY-BLACKWELL
Julkaisuvuosi: 2012
Journal: ChemBioChem
Tietokannassa oleva lehden nimi: CHEMBIOCHEM
Lehden akronyymi: CHEMBIOCHEM
Numero sarjassa: 1
Vuosikerta: 13
Numero: 1
Aloitussivu: 120
Lopetussivu: 128
Sivujen määrä: 9
ISSN: 1439-4227
DOI: https://doi.org/10.1002/cbic.201100637
Tiivistelmä
Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C?C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C?C bond intact (nogalamycin R) or by 2-deoxyfucose without the C?C bond (nogalamycin F). In addition, a monoglycosylated intermediate3',4'-demethoxynogalose-1-hydroxynogalamycinonewas isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the clustersnogZappears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II.
Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C?C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C?C bond intact (nogalamycin R) or by 2-deoxyfucose without the C?C bond (nogalamycin F). In addition, a monoglycosylated intermediate3',4'-demethoxynogalose-1-hydroxynogalamycinonewas isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the clustersnogZappears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II.
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