Identification of a Cyclase Gene Dictating the C-9 Stereochemistry of Anthracyclines from Streptomyces nogalater

Abstract

Nogalamycin is an anthracycline antibiotic produced by Streptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.Most anthracyclines that have been investigated are biosynthesized via aklavinone as a key intermediate. Biosynthesis has been well demonstrated with daunomycins (e.g., see references 8 and 13), which, due to their clinical significance, are the best known anthracyclines. However, nogalamycin (Fig. 1A), produced by Streptomyces nogalater (ATCC 27451), is a distinctive anthracycline. Its aglycone (nogalamycinone) is synthesized via a polyketide biosynthetic pathway from 10 acetates (17). Nogalamycin has two sugar residues: a neutral sugar, nogalose, and a dimethyl amino sugar, nogalamine. The features that make nogalamycin different from most other anthracyclines are the attachment of nogalamine at both C-1 (by a typical glycosidic bond) and at C-2 (by an unusual C-C bond) (16) and the opposite stereochemistry at C-9 (1). In addition to the nogalamycin group, steffimycins are the only anthracyclines known to have the 9S configuration (2).It has not been possible to produce nogalamycinone ( Fig. 1B), either (i) chemically, because nogalamine could not be removed from the aglycone (only the bisanhydro form of the aglycone was obtained by treatment with strong base at elevated temperatures) (16), or (ii) by genetic engineering, due to the lack of the cyclase determining the unique stereochemistry at C-9. In this paper, we report the production of nogalamycinone by genetically engineered Streptomyces lividans TK24, clarifying an important intermediate of nogalamycin biosynthesis.Strains, culture conditions, and DNA manipulation. Streptomyces strains were grown at 30°C in tryptone soya broth containing thiostrepton (50 g/ml) for preparation of plasmid DNA and in E1 medium containing glucose (20 g/liter), soluble starch (20 g/liter), Farmamedia (5 g/liter), yeast extract (2.5 g/liter), CaCO 3 (3 g/liter), NaCl (1 g/liter), MgSO 4 ⅐ 7H 2 O (1 g/liter), and K 2 HPO 4 ⅐ 3H 2 O (1 g/liter) in tap water (pH 7.5) for production of anthracycline metabolites (19). A DNA fragment derived from a nogalamycin biosynthetic cluster containing a cyclase gene was cloned in a pIJ486-based plasmid and introduced into S. lividans TK24 (6). Streptomyces galilaeus mutant H039 producing aklavinone-(rhodinose) 2-3 (19) was used as a host in attempts to produce C-9 stereoisomers of aklavinone. DNA isolation and manipulation were carried out by standard methods (6, 14).Cloning and sequencing of the gene for nogalamycin cyclization. In our attempts to clarify the molecular genetics of nogalamycin biosynthesis, we expanded the previously characterized nogalamycin gene cluster (15,20,21). A fragment from this previously cloned nogalamycin biosynthetic...

In vitro characterization of IroB, a pathogen-associated C -glycosyltransferase

“…The five genes of the iroA cluster, iroB, iroC, iroD, iroE, and iroN, have been shown previously to be necessary and sufficient for (i) glucosylation of Ent, (ii) export of apo glucosyl-Ent, and (iii) import of holo (Fe III -bound) glucosyl-Ent (17,18). The protein encoded by the iroB gene is homologous to glycosyltransferases (Gtfs) from the nogalamycin (27) and erythromycin (28) gene clusters. To characterize the enzymatic C-glycosylation of Ent, we isolated genomic DNA from E. coli CFT073, a uropathogen containing the iroA gene cluster (21).…”

“…Very few examples of C-glycoside-bearing natural products have been described; among these are the maysinfamily maize flavones (36) and the polyketides granaticin (37), simocyclinone D8 (38), and the angucycline antibiotics (27,(39)(40)(41). All of the C-Gtfs must generate a carbon nucleophile to attack C1Ј of the glycosyl moiety of the donor cosubstrate NDP-hexose (Fig.…”

Engineering Anthracycline Biosynthesis toward Angucyclines

“…The biosynthesis of aklavinone, the aglycone moiety of aclacinomycin, progresses highly similarly to that of nogalamycin, with the exception of the use of a propionate starter unit, which results in an ethyl side chain in the fourth ring (14), and the stereochemistry in the cyclization of the fourth ring, which is opposite in the nogalamycin and aclacinomycin biosynthesis pathways (16). In this study, the differences in anthracycline starter unit utilization could be used to investigate the substrate flexibility of PgaF, as priming of the minPKS complex with propionate results in an unnatural substrate for PgaF that is composed of a longer carbon chain (Fig.…”

Structure of a conserved hypothetical protein, TTHA0849 fromThermus thermophilusHB8, at 2.4 Å resolution: a putative member of the StAR-related lipid-transfer (START) domain superfamily

“…TTHA0849 from Thermus thermophilus HB8 is a conserved hypothetical protein composed of 147 amino acids (M r = 17 084) and exhibits weak sequence identity (about 20%) to polyketide (oligoketide) cyclases that bind multicyclic and/or aromatic compounds (Dickens et al, 1995;Kendrew et al, 1999;Madduri & Hutchinson, 1995;Niemi & Mä ntsä lä , 1995;Rä ty et al, 2002;Torkkell et al, 2000). Based on the sequence similarity between TTHA0849 and a polyketide cyclase, SnoaL from Streptomyces nogalater, the folding of the polypeptide backbone and the active-site environment in the two proteins are expected to be conserved (Sultana et al, 2004).…”