Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

Abstract

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity towards a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4′-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogs. In this study, we identified, sequenced and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant groups addition is C-9 glycosylation, C-4 salicylation and O-4′ angelycylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity towards substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group towards structural-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases, C-6 methyltransferase and C-4/C-12a dihydroxylase were functionally reconstituted. Keywordstopoisomerase; polyketide; salicylate; biosynthesis; acyltransferase yitang@ucla.edu. Supporting Information Available: Additional Experimental procedures, and compound characterizations. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2010 December 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBacterial aromatic polyketide natural products comprise a group of molecules that displays diverse structures and bioactivities yet share common biosynthetic origins. The poly-β-ketone backbone is synthesized by a minimal polyketide synthase (PKS) consisting of a ketosynt...

The TetR Family of Regulators

“…This raises questions as to the possible origins of other TFRs, for example, TetR and MphR of E. coli, in producing organisms. No TFR has been identified in the biosynthesis clusters for oxytetracycline or chlorotetracycline, but a TFR is present in the cluster for the glycosylated anticancer tetracycline SF2575, although our analysis does not show a close relationship to TetR from E. coli (25,99). The closest homolog of MphR is, however, found in the environmental microbe Myxococcus xanthus (25,59), revealing possible origins for MphR in the environment.…”

Oxytetracycline Biosynthesis

“…Also present in the ssf gene cluster is an OxyI homolog, SsfY4, which was also determined to be unnecessary for cyclization of the tetracyclic scaffold, confirming the result seen with OxyI. More importantly, a putative acyl-CoA ligase, SsfL2, was required to produce the tetracyclic intermediate 12 in a heterologous host (61). A possible role of SsfL2 may be to catalyze the Claisen-like cyclization between C1 and C18 of hydrolyzed tricyclic anthracene carboxylic acids in a CoA-dependent fashion.…”

“…The recently sequenced gene cluster responsible for biosynthesis of the tetracycline-like compound SF2575 allowed reconstitution of the cyclization events in this pathway as well (61). The second ring cyclase in the ssf pathway, SsfY2, has low homology to OxyN while bearing strong homology to second ring cyclases from benzoisochromanequinone pathways such as Gra-ORF33 (60).…”

“…Alternatively, the enzyme responsible for this step in the CTC gene cluster contains an inactivating mutation. For additional comparison, the ssf gene cluster contains only two oxygenases, an OxyS homolog, SsfO1, and an OxyL homolog, SsfO2 (33,61). Therefore, the role of the two additional oxygenases and the enzymatic basis of the C5 hydroxyl remain an unsolved mystery of OTC biosynthesis.…”

Substrate specificity of benzamide synthetase involved in 4-hydroxy-3-nitrosobenzamide biosynthesis

“…5 OxyD and SsfD are involved in the biosynthesis of oxytetracycline in Streptomyces rimosus 6 and tetracycline SF2575 in Streptomyces sp. SF2575, 7 respectively. PcsA and PcsB are polyene carboxamide synthases in the polyene amide biosynthesis pathway in Streptomyces diastaticus 8 and the AB-400 biosynthetic pathway in Streptomyces RGU5.3, 9 respectively.…”