Modification of aklavinone and aclacinomycins in vitro and in vivo by rhodomycin biosynthesis gene products

Abstract

The rdm genes B, C and E from Streptomyces purpurascens encode enzymes that tailor aklavinone and aclacinomycins. We report that in addition to hydroxylation of aklavinone to ?-rhodomycinone, RdmE (aklavinone-11-hydroxylase) hydroxylated 11-deoxy-β-rhodomycinone to β-rhodomycinone both in vivo and in vitro. 15-Demethoxyaklavinone and decarbomethoxyaklavinone did not serve as substrates. RdmC (aclacinomycin methyl esterase) converted aclacinomycin T (AcmT) to 15-demethoxyaclacinomycin T, which was in turn converted to 10-decarbomethoxyaclacinomycin T and then to rhodomycin B by RdmB (aclacinomycin-10-hydroxylase). RdmC and RdmB were most active on AcmT, the one-sugar derivative, with their activity decreasing by 70–90% on two- and three-sugar aclacinomycins. Aclacinomycin A competitively inhibited the AcmT modifications at C-10. The results presented here suggest that in vivo the modifications at C-10 take place principally after addition of the first sugar.

Anthracycline, Aclacinomycin, Rhodomycin, rdm gene, Streptomyces

1 Introduction

Anthracyclines such as daunorubicin, doxorubicin and aclacinomycin belong to a class of antibiotics produced by Streptomyces bacteria exhibiting extraordinary cytotoxic effects [1,,3]. Biosynthetic and structural studies have shown that formation of carminomycin, daunorubicin and doxorubicin requires hydroxylation at C-11 of the key precursor aklavinone (Akv) [4,,,7]. This hydroxylation occurs prior to glycosylation, with further modifications also taking place after glycosylation to produce structurally related derivatives. Fujii and Ebizuka [8] have summarized the biosynthesis of aclacinomycins and Akv produced by Streptomyces galilaeus.

A new anthracycline hybrid was produced by S. galilaeus following the introduction of rhodomycin biosynthesis genes from Streptomyces purpurascens[6,9,10]. The rdm ABCDEF gene cluster from S. purpurascens encodes the enzymes responsible for the 11- and 10-modifications of aklavinone and its glycosides [6]. RdmE is an aklavinone-11-hydroxylase which catalyzes the formation of ?-rhodomycinone from Akv (Fig. 1) [7,10]. RdmC catalyzes the removal of the methyl group from C-15 of aclacinomycin T (AcmT) [9]. RdmB decarboxylates the C-10 carboxylic acid and subsequently adds an hydroxyl group at C-10 of AcmT [9]. However, the molecular mechanisms underlying the 10-modification catalyzed by RdmB and correlation of glycosylation events and tailoring of the aglycone moiety are still unclear.

1

Modifications of aclacinomycins and their aglycones produced by various S. galilaeus strains and H0 mutants applied to study the specificity of RdmC, RdmB and RdmE. The structures have been described earlier [11,12].

The present investigation sought to determine the substrate specificities of RdmE, RdmC and RdmB by using different aclacinomycins or their aglycones in both in vivo and in vitro studies. The data show that in the two sequential reactions catalyzed by RdmC and B, the preferred substrate was AcmT, which contains one sugar unit, Rhn. AcmA and other three- or two-sugar aclacinomycins competitively inhibited C-10 modifications in AcmT.

2 Materials and methods

2.1 Bacterial strains, plasmids and culture conditions

The strains and plasmids used in this study are listed in Table 1. The rdmB, rdmC, rdmD or rdmE genes, and their different combinations, were introduced into the expression vector pIJ487, and were under the control of the ermE or the rdmE promoter [7,9]. Streptomyces strains were grown for 4–7 days at 30°C on ISP4 agar plates for spore preparation. The protoplasts from Streptomyces lividans and S. galilaeus were prepared with 100 μl spore suspension or with spores taken from ISP4 agar plates added to 25 ml YEME medium [15] and to 25 ml SGYEME medium [15], respectively. To obtain anthracyclines, the strains were cultivated in E1 medium [10]. For plasmid-containing strains, tsr was added to 5 μg ml⁻¹. S. lividans TK 24 carrying pRDM plasmids was grown in TSB medium (Oxoid Tryptone Soya Broth powder, Difco) at 30°C for 3–4 days. Escherichia coli XL-2 blue carrying pRDM16 was cultured in Luria-Bertani broth containing 100 μg ml⁻¹ ampicillin [16]. Plasmid DNA preparations isolated from S. lividans TK 24 were always first introduced into the H039 mutant which is more easily transformed than the other S. galilaeus mutants. Plasmids isolated from H039 were further introduced into the other H0 mutants.

1

Bacterial strains and plasmids used in this study

Bacterial strain and plasmid	Characteristics	Reference
S. purpurascens ATCC 25489	donor of rdm genes	[10]
S. galilaeus ATCC 31615	producer of Acm A	[13]
S. galilaeus H026	producer of Acm N	[14]
S. galilaeus H038	producer of Acm T	[14]
S. galilaeus H039	producer of Acm I	[14]
S. galilaeus H054	producer of Acm H	[14]
S. galilaeus H075	producer of Acm M	this work
S. lividans TK24	non-producing strain, cloning host	[15]
E. coli XL-2 blue	cloning host	Stratagene
pRDM6	rdmABCDEFa	[6]
pRDM10	rdmEa	[7]
pRDME*12	rdmCb	[9]
pRDM13	rdmBCa	[9]
pRDM14	rdmBCDa	[9]
pRDM16	rdmB in pGEX 4T-3c	[9]

^aermE promoter

^brdmE promoter

^ctac promoter

2.2 Production and detection of anthracyclines and their aglycones

S. galilaeus ATCC 31615 was transformed with pRDM10 and pRDM6, and S. galilaeus mutants producing different aclacinomycins with pRDME*12 and pRDM13. To screen novel products, the transformed strains were incubated in E1 medium for 5–7 days. The glycosides and aglycones were isolated and detected as previously described [7,10].

2.3 Biotransformation

Anthracyclines (approximately 1–2 mg) and their aglycones (approximately 100 μg) were dissolved in 2% methanol and added to 10 ml of Streptomyces cultures in E1 medium. The samples (1 ml) were removed after 1 h biotransformation and extracted with 600 μl of methanol/toluene/1 M sodium phosphate (pH 7.0) mixture (1:1:1 by volume). Detection of glycosides and their aglycones was as previously described [7,10].

2.4 Purification of RdmE, RdmC and RdmB and enzyme assay

Purification of RdmE from S. lividans TK24 carrying pRDM10 was as previously described [7] except the Sepharose Cl-2B step was omitted. RdmB was purified as a GST fusion protein according to the supplier's instructions (Amersham-Pharmacia) and as described previously [9] from E. coli XL2-blue (Stratagene) carrying pRDM16. RdmC was purified from S. lividans TK24 carrying pRDME*12[9].

RdmE was assayed as previously described [7]. The reaction was followed kinetically at 500 nm. Methanol concentration was kept below 2%. RdmC and RdmB were assayed as previously described [9]. Acm concentration in the assay varied from 4 to 86 μM.

2.5 In vitro modifications of the polyketide skeleton in aclacinomycins

Aclacinomycins (36–86 μM) were incubated with purified RdmC or with the combination of RdmC and RdmB (approximately 10 μg enzyme) at 37°C for 30 min. After addition of a 1:1 (v/v) mixture of 0.1 M HCl and toluene, glycosides were hydrolyzed with refluxing and mixing at 85°C for 30 min. The extracted aglycones were analyzed by TLC on silica gel F₂₅₄ plates [10].

3 Results

3.1 Specificity of C-11 and C-10 modifications in vivo

S. galilaeus H038/pRDME*12 and S. galilaeus H038/pRDM13 cultures contained complex mixtures of products when analyzed by 2-dimensional thin layer chromatography (TLC). However, the main product was AcmT (roughly 40% of the total amount of aclacinomycins). When the mixtures were hydrolyzed as described above (Section 2.5) and the aglycones were fractionated by a silica column the main products were 15-dmAkv and 11-deoxy-β-rhodomycinone, respectively. The next experiments investigated whether the C-10 tailoring enzymes RdmC and RdmB acted preferentially on AcmT or whether the two- and three-sugar aclacinomycins were equally active as substrates. pRDME*12 and pRDM13 were introduced into S. galilaeus mutants producing Acm with one, two or three sugar residues. The plasmids pRDM10 and pRDM6 were introduced into S. galilaeus ATCC 31615, which produces AcmA as the main product. Table 1 lists the various aclacinomycins made by S. galilaeus ATCC 31615 and by the H0 mutants, while Fig. 1 shows the structures before and after modification by the products of the introduced rdm genes. The strains carrying pRDME*12, pRDM13, pRDM10 or pRDM6 were grown in E1 medium at 30°C for 5–7 days. S. galilaeus ATCC 31615 and the mutants H026, H038, H039, H054 and H075 carrying pRDME*12, all produced 15-dmAkv as the main product after hydrolysis. The amounts of 15-dmAkv produced by H038 were roughly 2-fold compared to the amounts produced by the mutants H026, H075 and the ATCC 31615 type strain. The amounts produced by the mutants H039 and H054 were even lower (1/3 to 1/4 of that produced by H038). The main product from the mutants carrying pRDM13 after hydrolysis was 11-deoxy-β-rhodomycinone. The new product from the wild-type S. galilaeus carrying pRDM10 was ?-rhodomycinone, while from S. galilaeus/pRDM6 the new products were ?-rhodomycinone and β-rhodomycinone (ratio 40:60). The mutants H026, H039, H054 and H075 produced three-sugar aclacinomycin derivatives, while H038 produced the one-sugar derivative, AcmT [10]. After hydrolysis, the only detected aglycone was aklavinone

3.2 Biotransformation of anthracyclines by S. lividans and S. galilaeus strains

S. lividans carrying pRDM6 or pRDM14 caused biotransformation of AcmT to 10-dcmAcmT and rhodomycin B within 1 h of reaction time. Biotransformation of AcmA in S. lividans carrying pRDM6 or pRDM14 caused similar C-10 modifications. However, the detected amounts of 10-dcmAcmA and rhodomycin B after 1 h were roughly 1/3 of biotransformation products of AcmT. AcmT was converted to rhodomycin B in S. galilaeus ATCC 31615 and in H038 transformed with pRDM6, pRDM13 or pRDM14. Only small amounts of 10-dcmAcmT were detected after 1-h incubation time. Based on hydrolysis of the glycosides, the ratio of 10-decarbomethoxyl and 10-hydroxyl derivatives after 1 h biotransformation of AcmA was roughly 1:1. Control incubations showed that AcmT was not converted to any product in H038 transformed with pIJ487.

3.3 Modification of the aklavinone skeleton at C-11 and C-10 in vitro

Purified RdmE modified Akv and 11-deoxy-β-rhodomycinone at C-11 and produced ?-rhodomycinone and β-rhodomycinone, respectively (Table 2), with 11-deoxy-β-rhodomycinone being the better substrate. The results suggest that replacement of the carbomethoxyl group with the hydroxyl group at C-10 favors the addition of an 11-hydroxyl group in the polyketide backbone. On the other hand, the carboxyl group at C-10 completely abolishes the 11-hydroxylase activity. Acm with one or three sugar residues functions as the substrate for RdmC and RdmB in vitro. However, Acm with one sugar, e.g. AcmT, served as the primary substrate (Table 2).

2

Activity of RdmE (a and of RdmC (b with different substrates

Substrate	Product	Activity (units mg−1)
a
Aklavinone	ɛ-rhodomycinone	70.6a
11-deoxy-β-rhodomycinone	β-rhodomycinone	118.6a
Decarbomethoxyaklavinone		0a
15-demethoxyaklavinone		0a
b
Aklavinone		0b
Acm T	15-demethoxyaclacinomycin T	184.0b
Acm A	15-demethoxyaclacinomycin A	51.0b
Acm N	15-demethoxyaclacinomycin N	59.6b
Acm M	15-demethoxyaclacinomycin M	46.0b
Acm H	15-demethoxyaclacinomycin H	8.8b
Acm I	15-demethoxyaclacinomycin I	25.5b

Aglycone and aclacinomycin concentrations in the reaction mixture were 40 μM and 36 μM, respectively. To measure initial velocities, about 10 μg purified enzyme was used.

^a1 unit converted 1 μmol aklavinone min⁻¹.

^b1 unit concerted 1 μmol AcmT min⁻¹.

3.4 Specificity of in vitro modifications

The data presented in Table 2 show that all tested aclacinomycins served as substrates for RdmC in vitro, although the production of 15-demethoxyaclacinomycin T from the three-sugar aclacinomycin derivatives was much lower than from AcmT. The data suggest that RdmC preferentially modifies the polyketide skeleton after addition of the first sugar residue. In order to test this hypothesis we incubated RdmC in the presence of various amounts of AcmT and 83 μM and 166 μM AcmA. As shown in Fig. 2, AcmA competitively inhibited RdmC.

2

Inhibition of RdmC by aclacinomycin A. Enzyme activity was determined as described under Section 2. Symbols are (?) no added AcmA; (•) 83 μM AcmA added; (▴) 166 μM AcmA added. Data were plotted using Lineweaver–Burke equation.

4 Discussion

Aklavinone is an important intermediate in the daunorubicin/doxorubicin and aclacinomycin biosynthesis pathways. Aklavinone undergoes several successive chemical modifications such as hydroxylation, glycosylation, oxidation and methylation [5,,,,9]. DnrF from Streptomyces peucetius and RdmE from S. purpurascens catalyze the first step among these modifications, i.e. the 11-hydroxylation of Akv to ?-rhodomycinone. Although DnrF acts early in the biosynthetic pathway of daunorubicin it does not mean that 11-hydroxylation is required for all the successive modifications to take place. A dnrF negative S. peucetius 7600 mutant (a yellow mutant) is able to produce 11-deoxydaunorubicin and 11-deoxydoxorubicin [17]. Similarly, production of aglycones with 10-modifications was achieved by using gene constructions lacking rdmE[6]. This supports an alternative hypothesis that the temporal order is simply linked to the substrate specificity of the enzymes because DnrF and RdmE utilize the 11-deoxy compounds downstream of Akv in the biosynthetic pathway much less efficiently than Akv. In particular, the enzymes were completely inactive on the C-7 glycosylated intermediates [5,9]. The hydroxyl group at C-11 of anthracyclines seems to have a primary biological role, since the red compounds (11-hydroxylated) are more toxic for Streptomyces itself and more cytotoxic for animals than the yellow 11-deoxy analogues [11].

The results showed that RdmE might function only before glycosylation in the rhodomycin biosynthesis pathway; it modified the aglycones to ?-rhodomycinone, 10-deoxy-β-rhodomycinone and β-rhodomycinone. After C-11 modification, successive tailoring steps such as glycosylation, 15-demethoxylation, 10-decarboxylation and 10-hydroxylation can take place. Here we have shown that when rdmC and rdmBC were introduced into H054 (the main product being Akv-dF-dF-CinA), only a small amount of new product was observed whereas in the wild-type S. galilaeus ATCC31615, RdmC and RdmB were able to modify the three-sugar derivative where an aminosugar, Rhn, is the first residue. These results suggest that rdmC and rdmBC poorly modify Akv when the neutral sugar dF or Rho is the first sugar residue. Similarly, in vivo the enzymes less effectively tailor aclacinomycins with a neutral sugar as the first residue. Purified RdmC alone, or purified RdmC and RdmB together, tailor effectively aglycones carrying one appropriate sugar residue, preferably an aminosugar. Dickens et al [18] reported that one-sugar daunorubicin derivatives act as the important substrates in the conversion of carminomycin and 13-dihydrocarminomycin to daunorubicin and 13-dihydrodaunorubicin, respectively, and also in the modification of daunorubicin to doxorubicin. Substrate specificity of the tailoring enzymes in vitro does not always correlate with the activities in vivo. Both 13-dihydrocarminomycin and carminomycin are good substrates for 4-O-methyltransferase in vitro. However, 13-dihydrodaunomycin, which is the product of the 4-O-methylation of 13-dihydrocarminomycin, is not a precursor to daunorubicin. This will rule out 13-dihydrocarminomycin as a substrate for 4-O-methyltransferase in vivo [19].

The present findings lead us to conclude that during the biosynthesis of β-rhodomycins, RdmE first modifies Akv at C-11, and ?-rhodomycinone or Akv is subsequently glycosylated. After the appropriate first sugar has been linked to the aglycone, RdmC and RdmB perform modifications at C-10.

Acknowledgements

The authors gratefully acknowledge financial support from the Academy of Finland.

Abbreviation

Abbreviation
• tsr

  thiostrepton

• Acm

  aclacinomycin

• 15-dmAcm

  15-demethoxyaclacinomycin

• 10-dcmAcm(T)

  10-decarbomethoxyaclacinomycin(T)

• Akv

  aklavinone

• dmAkv

  demethoxyaklavinone

• dcmAkv

  decarbomethoxyaklavinone

• Rhn

  rhodosamine

• Rho

  rhodinose

• dF

  2-deoxy-L-fucose

• CinA

  L-cinerulose A

• RdmE

  aklavinone-11-hydroxylase

• RdmC

  aclacinomycin methyl esterase

• RdmB

  aclacinomycin-10-hydroxylase