Actinorhodin (ACT, 1), an aromatic polyketide produced by Streptomyces coelicolor A3(2),1 belongs to a class of benzoisochromanequinone (BIQ) antibiotics (Figure 1a).2 Because 1 has a symmetrical structure in which two BIQ units are coupled via a C–C bond, its enzymatic formation in a regiospecific manner has drawn considerable attention. ACT biosynthesis is controlled by the act cluster, which comprises 22 genes, and the functions of several genes remain elusive. To characterize one such gene, the deletion mutant of actVA-ORF4 (ΔactVA-4) was constructed (Supplementary Figure S1). Analysis of ACT-related metabolites produced by this mutant led to the identification of two characteristic compounds: (1R,3S)-6,9-dihydroxy-1-methyl-5,10-dioxo-3,4,5,10-tetrahydro-1H-naphtho-[2,3-c]-pyran-3-ylacetic acid, known as 8-hydroxy-dihydrokalafungin (DHK-OH, 2) and (1R,3S)-5,10-dihydroxy-1-methyl-6,9-dioxo3,4,6,7,8,9-hexahydro-1H-naphto-[2,3-c]-pyran-3-ylacetic acid, known as 8-hydroxy-3,4,7,8-tetrahydrokalafungin (THK-OH, 3; Figure 1a).3 The structures of 2 and 3 correspond to the monomeric unit of 1, thus strongly suggesting actVA-4 to be an essential component for dimerization at C-10 of ACT biosynthesis. Further metabolite analysis of the ΔactVA-4 culture broth revealed the presence of a third unidentified ACT-related metabolite (compound Z; Figure 1b), which is absent in the wild-type broth. This turned out to be a new shunt product of ACT biosynthesis; the structure elucidation and biosynthetic implication of this new product are presented in this paper.

Figure 1
figure 1

The proposed biosynthetic pathway of ACT and structural elucidation of 5. (a) The biosynthetic relationship of the metabolites mentioned in this study. (b) HPLC profiles of ethyl acetate extracts from liquid culture of wild type (top) and ΔactVA-4 (bottom) under the following conditions: TOSOH TSK-gel ODS-100 V, 4.6 mm i. d. × 150 mm, 40 °C, 10–95% aqueous acetonitrile containing 0.5% acetic acid, 1.0 ml min−1. Chromatograms were monitored by absorbance at 254 nm. (c) Structure of 5. Numbering of positions is based on the biosynthetic order. (d) Correlations of COSY (bold line) and HMBC (arrow).

Full size image

The ΔactVA-4 mutant was inoculated into tryptic soy broth medium for seed culture4 and grown on a rotary shaker at 200 r.p.m. and 30 °C for 2 days. Aliquots of the seed culture were transferred to R5MS liquid medium and grown as described previously.5 HPLC analysis of the culture broth showed that the UV-VIS spectrum of compound Z is similar to that of 5,14-epoxy-kalafungin (Supplementary Figure S2).6 LC/HRESIMS provided a molecular formula of C21H23NO10S (m/z [M-H]− calcd for C21H22NO10S, 480.0964. found, 480.0977), suggesting the attachment of a moiety containing nitrogen and sulfur atoms. Subsequently, the ethyl acetate (EtOAc) extract obtained from the large-scale culture (6.6 l) was subjected to silica-gel column chromatography eluting with EtOAc/hexane (6:4, 4:6 and 2:8, stepwise) and acetone/methanol (MeOH; 9:1). The fraction containing compound Z was subsequently subjected to preparative HPLC twice (TOSOH TSK-gel ODS-100 V, 7.8 × 300 mm, 50 °C, solvent A: water containing 0.01% TFA, solvent B: acetonitrile containing 0.01% TFA. First preparation: 25% B isocratic, 2.0 ml min−1. Second preparation: 20% B isocratic, 2.5 ml min−1) to yield a pure compound (8.2 mg).

Compound Z was isolated as a white amorphous solid: [α]D23+116° (c 0.10, MeOH); UV λmax nm (log ɛ) in MeOH: 208 (4.31), 232 (4.23), 260 (sh, 4.00), 354 (3.70); IR (ATR) νmax cm−1 3383 (hydroxyl), 1692 and 1651 (carbonyl). NMR data of compound Z in dimethylsulfoxide (DMSO)-d6 indicated the existence of 21 carbons and at least 19 protons (Table 1). Three doublet–doublet signals at 7.29, 7.43 and 7.73 p.p.m. in 1H-NMR and their HMBC correlations indicated a 2,3-dihydro-8-hydroxy-1,4-naphthoquinone moiety. Further comparison of the NMR data between compound Z and dihydrokalafungin (DHK, 4, Figure 1a) strongly suggested the presence of a pyran ring moiety with a carboxyl group. Compound Z seemed to be a derivative of 4; however, the signals assigned to C-5 (δC 58.6) and C-14 (δC 76.0) indicated attachment of hetero atoms to C-5 and C-14, respectively, and a single bond connection between C-5 and C-14. The COSY spectrum indicated the connectivity of the methylene peak (δC 31.7; δH 2.46, 2.57) with methine (δC 51.2; δH 4.14), which in turn correlated with the NH proton (δH 8.12; Figure 1d). These data, as well as the additional signals derived from the acetyl (δC 22.3, 169.3; δH 1.75) and carboxyl (δC 171.4) groups, indicated the existence of an N-acetyl cysteine (AcCys) moiety. The key HMBC correlation could be successfully detected between the methylene proton (δH 2.46, 2.57) and C-5 (δC 58.6), thus clearly indicating that the AcCys moiety attaches to C-5 via a sulfur atom.

Table 1 NMR data of 5R-AcCys-14S-OH-DHK, 5, in DMSO-d6
Full size table

The MS data indicated the presence of an oxygen atom in addition to the aforementioned structure, and the attachment of hydroxyl group at C-14 was reasonably confirmed on the basis of the HMBC correlation from the proton signal at 7.10 p.p.m. to C-5 (δC 58.6). Considering the assumption of the biosynthetic relationship with ACT, the structure of compound Z was elucidated as 5-(N-acetyl-L-cysteinyl)-14-hydroxy-dihydrokalafungin (5-AcCys-14-OH-DHK, Figures 1c and d). In 2D rotating frame NOESY (ROESY) analysis (Supplementary Figure S3), ROEs between 14-OH and H-4α, and between 14-OH and H-15 suggested the C-14 stereochemistry as (S)-configuration. Similarly, ROEs between H-16 and H-3, between H-3 and H-4β, and between H-4β and H-3’ indicated the C-5 stereochemistry as (R)-configuration. The absolute structure of the compound was thus elucidated as 5R-(N-acetyl-L-cysteinyl)-14S-hydroxy-dihydrokalafungin (5R-AcCys-14S-OH-DHK, 5; Figure 1c), which is (1R,3R,4aR,10aS)-4,4a,5,10,10a-hexahydro-9,10a-dihydroxy-5,10-dioxo-1-propyl-4a-[[(2R)-2-(acetylamino)-2-carboxyethyl]thio]-1H-Naphtho[2,3-c]pyran-3-acetic acid.

Certain Gram-positive bacteria, including streptomycetes, use mycothiol (MSH, 6) to protect the cells against toxic and/or reactive electrophiles. MSH has analogous functions to glutathione and can react with various toxic compounds to form MSH S-conjugates, which are then cleaved by an MSH S-conjugate amidase to release GlcN-Ins and a toxin-AcCys S-conjugate.7, 8 The metabolic genes for MSH are present in S. coelicolor A3(2),9 and two AcCys adducts of 4 were identified from a recombinant S. coelicolor strain.10 ActVA-ORF5 is a bifunctional flavin-dependent monooxygenase that catalyzes the oxygenation reactions at both the central (C-6) and lateral (C-8) rings of a tricyclic substrate, 6-deoxy-dihydrokalufungin (DDHK, 7).9 Formation of 5 apparently occurred via the ring-opening reaction of a 5,14-epoxy intermediate, which is most plausibly the 5,14-epoxide of a trihydroxynaphthalene (T3HN) derivative (8; Figure 1a). A hydroquinone moiety of 8 was unstable and could be easily oxidized to an isolable quinone form. Although there is no report on epoxide derivatives of 1 from S. coelicolor strains, diepoxyactinorhodin has been isolated from a different Stretpomyces strain.11 Another recent example related to 5 is the isolation of frenolicin C with the same substitution pattern of hydroxyl and AcCys groups but with a different carbon chain length of polyketide origin.12