Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5

Abstract

(4,5,8,9,33), a pathway in which two methyltransferase reactions have been proposed (8,9,33). Aklanonic acid methyltransferase (AAMT) catalyzes the methyl esterification of aklanonic acid (Fig. 1A) (8). Aklanonic acid methyl ester (AAME) cyclase catalyzes the formation of aklaviketone from AAME, and in wild-type anthracycline producers, aklaviketone is reduced to form aklavinone (8). We have generated mutants specifically blocked in AAMT activity (dauC mutants) and AAME cyclase activity (dauD) (4, 5).Carminomycin 4-O-methyltransferase (CMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 4-O-position of carminomycin and 13-dihydrocarminomycin (Fig. 1B) to form daunomycin and 13-dihydrodaunomycin, respectively (9, 10). CMT has been purified to near homogeneity and is an apparent homotetramer with an M r of ca. 161,000 (10). A gene from S. peucetius ATCC 29050 encoding CMT has been isolated and sequenced (25). In this article, we show the isolation and sequence analyses of AAMT, AAME cyclase, and CMT of Streptomyces sp. strain C5. On the basis of a comparison of the sequence similarities and differences, the two methyltransferases apparently belong to two different subgroups. The AAME cyclase appears to be unusual with respect to both its activity and proteins in the databases. We also show the sequence of dauP, encoding an esterase-like function which we hypothesize also to be involved in daunomycin biosynthesis. MATERIALS AND METHODSBacterial strains, plasmids, and media. Streptomyces sp. strain C5 and mutants derived from it have been described previously (4, 5). Streptomyces lividans TK24 (18) was obtained from D. A. Hopwood. Streptomyces strains normally were grown in YEME (18) supplemented with 20% sucrose. R2YE medium, also used for growth as well as for preparation of streptomycete protoplasts, was prepared as described by Hopwood et al. (18). Nitrate-defined-plus-yeast-extract (NDYE) medium, used for growth of Streptomyces sp. strain C5 and its mutants, has been described previously (9). Strains carrying streptomycete plasmids pIJ486 (38), pIJ702 (18), and pWHM3 (36) or derivatives of them were grown and stored on plates containing 40 g of thiostrepton per ml.Escherichia coli JM83 was used to propagate plasmids for sequencing and restriction analyses. E. coli was grown in Luria-Bertani medium, and plasmids were introduced into E. coli by transformation done by standard procedures (26). For E. coli strains harboring plasmids, ampicillin was added at a final concentration of 100 g/ml.General genetic manipulations. The procedures for protoplast formation, transformation, and regeneration of protoplasts for Streptomyces sp. strain C5 have been described elsewhere (24). The procedures used for the preparation of Streptomyces plasmid and chromosomal DNA have been described by Hopwood et al. (18). The digestion of DNA with restriction endonucleases was carried out according to the manufacturers' directions. Restriction mapping and other routine molecular methods used in th...

Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus

“…Furthermore, ORF10 and ORF12 could be responsible for the introduction of the methoxyl group onto the indole ring. ORF12 shows sequence similarity to cytochrome P450 enzymes which catalyse the hydroxylation of flavonoids (Ueyama et al, 2002), while ORF10 is a homologue of methyltransferases which catalyse the methylation of a phenolic hydroxyl group (Dickens et al, 1995). By sequence analysis, however, it is not possible to assign the prenyltransferases FtmPT1 and FtmPT2 to the corresponding reactions.…”

Crystal Structure of Aclacinomycin Methylesterase with Bound Product Analogues

“…The polypeptide chain contains 298 amino acids, and the enzyme is monomeric in solution (14). RdmC shows about 50% sequence identity to related enzymes from other Streptomyces species, for instance DnrP from Streptomyces peucetius and DauP from Streptomyces sp strain C5 (17,18). These enzymes catalyze similar reactions, hydrolyzing 10-carbomethoxy-13-deoxycarminomycin to 10-carboxy-13-deoxycarminomycin (12,17).…”

“…C5 exhibit sequence identities to RdmC of 51 and 53%, respectively (12,17). DnrP and DauP have both been proposed to act as 10-carbomethoxy-13-deoxycarminomycin esterases on anthracycline substrates removing the carboxymethyl side chain at the C-10 position of, for example, the rhodomycin D aglycones (DauP) (18). Rhodomycin D is one of the precursors in the production of daunorubicin and doxorubicin, both clinically important anthracycline chemotherapeutic agents.…”

“…These enzymes catalyze similar reactions, hydrolyzing 10-carbomethoxy-13-deoxycarminomycin to 10-carboxy-13-deoxycarminomycin (12,17). They all contain the same sequence motif G-X-S-X-G, typical for a serine hydrolase active site (18).…”

Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp. The GenBank accession number for acmA reported in this paper is AF043550.

“…1a) in aclacinomycin biosynthesis. acmA was similar (83 % similarity and 70 % identity at the amino acid level) to dauD encoding AAME cyclase for daunomycin biosynthesis (Dickens et al, 1995). The gene was inserted into pSY15 from the 3 kb BamHI fragment using restriction sites Eco47III and BamHI (Fig.…”