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 toward 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 analogues. 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-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. 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 toward 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 toward structure-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.

Figure 1

Production of 1 and biosynthetic intermediates by S. sp. SF2575. The data were collected on the Shimadzu LC-MS. (A) LC trace (358 nm) for a mixture of standards 6, 7 and 1. (B) LC trace (358 nm) of the extract after 7 days growth on solid Bennette's media. (C) Selected ion monitoring was used to confirm the identification of 7 ([M+H]⁺ m/z = 696) and 6 ([M+H]⁺ m/z = 576). The UV spectra of these compounds are shown for comparison.

Figure 2

Organization of the ssf biosynthetic gene cluster. Genes are categorized according to their proposed role. The gene cluster spans 47.2 kb and contains 40 ORFs. Details of proposed functions are shown in Table 1.

Figure 3

In vitro assay of SsfL1 activity and substrate specificity. (A) Synthesis of salicylyl-AMP as indicated by the release of PP_i in the presence of ATP, salicylate 19 and the salicylyl-CoA ligase SsfL1. i) all reaction components; ii) no 19; iii) no ATP; iv) no SsfL1; and v) pyrophosphate reagent only. (B) Utilization of substituted benzoic acids by SsfL1 as indicated by PP_i release assay. The structures of compounds indicated by number here are shown in Scheme 3.

Figure 4

In vitro assay of SsfX3 activity and substrate specificity. (A) The tandem actions of SsfL1 and SsfX3 transfer 19 to the aglycon substrate 6 to yield 7. The assays are performed in 50 mM HEPES, pH 7.9 and 10 mM MgCl₂. i) the semisynthetic 6 standard; ii) the semisynthetic 7 standard; iii) Complete reaction containing 50 mM HEPES pH 7.9, 10 mM MgCl₂, 2 mM ATP, 2 mM free CoA, 2 mM 19, 20 μM 6, 1.5 μM SsfX3 and 15 μM SsfL1. Control reactions were performed as iii) with the following exclusions: iv) no SsfL1; v) no ATP; vi) no 19; vii) no SsfX3; and viii) no CoA. The reactions were examined with HPLC (358 nm). (B) Synthesis of analogs of 7 using salicylic acid analogs, SsfL1 and SsfX3. All reactions were performed at 25 °C for 30 minutes, extracted with organic solvent and analyzed by LC-MS (358 nm, positive ionization).

Figure 5

Reconstitution of tetracycline intermediates using ssf genes expressed in S. lividans K4-114. (A) HPLC analysis (245 nm) of the K4-114/pLP27 extract shows the amidated, reduced polyketide 42 is the major product, confirming the biosynthesis of the polyketide backbone 40 by SsfABCD and the subsequent C-9* reduction by SsfU. (B) HPLC analysis (253 nm) of the K4-114/pLP27/pLP77 extract shows addition of the putative cyclase SsfY1 leads to complete cyclization and aromatization of the D and formation of the shunt benzopyrone 44. (C) HPLC analysis (430 nm) of K4-114/pLP27/pLP126 extract shows 50, the oxidized form of 49, as the dominant product. Biosynthesis of 49 using entirely ssf genes (SsfABCDUY1Y2M4L2) indicates the tetracycline nature of the ssf biosynthetic pathway. (D) HPLC analysis (395 nm) of K4-114/pLP36 extract confirms the function of SsfO2 as the oxygenase that dihydroxylated C-4 and C-12a of 49. The resulting product 51 undergoes spontaneous degradation to afford the observed product 52.

Scheme 1

Scheme 2

Scheme 3

Scheme 4