Identification of Late‐Stage Glycosylation Steps in the Biosynthetic Pathway of the Anthracycline Nogalamycin

Abstract

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 intermediate-3',4'-demethoxynogalose-1-hydroxynogalamycinone-was 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 cluster-snogZ-appears 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.

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

“…S1F, and Tables S1 and S2). The unexpected attachment of olivose in 4, 6, and 7 is likely caused by endogenous enzymes from the heterologous host S. albus, which have been noted to influence anthracycline glycosylation patterns (9).…”

“…1) (9), which contains the C5′′-C2 bond, but has (S) stereochemistry at C4′′, in contrast to the 4′′-(R) configuration seen in 1. The functions of most gene products residing in the gene cluster have already been mapped (1,9,14), which directed us toward two putative α-KG-and Fe(II)-dependent oxygenases, snoN and snoK, as the likely candidate genes for late stage tailoring reactions.…”

“…Isolation of plasmid and cosmid DNA from Streptomyces species was performed using conventional techniques (31). For protein production, the gene snoK was amplified from the cosmid pSnogaori and snoN from the plasmid pUCZOMLTN (9). The genes were cloned into a modified pBAD/HisB-plasmid (33).…”

“…However, despite intensive investigations into the biosynthesis of 1, the mechanism of carbocyclization has remained unknown (1). Furthermore, recent molecular genetic studies have revealed, unexpectedly, that the amino sugar attached at C1 is originally rhodosamine (9). The opposite stereochemistry at C4′′ of rhodosamine in comparison with nogalamine implies that a stereoinversion reaction must occur at a late stage in the biosynthesis.…”

Biosynthetic pathway toward carbohydrate-like moieties of alnumycins contains unusual steps for C-C bond formation and cleavage

“…For production of the metabolites, Streptomyces strains were routinely cultivated in 25 mL of NoS-E1soy medium (27) with added Amberlite XAD 7HP (Rohm and Haas) resin (1 g∕50 mL) for 5 d at 300 rpm, 301 K. MS plates (28) were used as a solid medium for S. albus (29). Apramycin at 50 μg∕mL and thiostrepton at 50 μg∕mL and 40 μg∕mL were used for selection in solid and liquid media, respectively.…”