1 Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA
2 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331-3507, USA
Correspondence
Taifo Mahmud
taifo.mahmud{at}oregonstate.edu
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ABSTRACT |
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Present address: Department of Chemical and Biological Engineering, Gyeongsang National University, Jinju 660-701, Korea.
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INTRODUCTION |
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Despite the preparation of a large number of rifamycin derivatives by semi-synthetic approaches, the structural modifications have been limited primarily to one region of the molecule, the C-3 or C-4 positions of the aromatic core unit (Cricchio et al., 1974, 1975
; Maggi et al., 1965
; Wehrli et al., 1987
). Alterations at other locations are difficult to accomplish chemically due to the complexity of the molecule, and require the implementation of alternative methodology, including combinatorial biosynthesis or mutasynthesis, to achieve additional structural diversity.
The biosynthesis of rifamycin has been studied extensively by both classical feeding and mutagenesis experiments, as well as by contemporary genetic and biochemical approaches (August et al., 1998; Floss & Yu, 2005
; Ghisalba & Nuesch, 1981
; Schupp et al., 1998
). The polyketide framework of rifamycin B is assembled from 3-amino-5-hydroxybenzoic acid (AHBA), which arises from the aminoshikimate pathway (Kim et al., 1998
; Yu et al., 2001
), two molecules of acetate and eight molecules of propionate. Synthesis of the core structure of rifamycin is catalysed by five multifunctional proteins (RifARifE) and an amide synthase (RifF) (Fig. 1A
) (August et al., 1998
; Schupp et al., 1998
). A loading module that contains domains homologous to adenylation and thiolation (A-T) domains of non-ribosomal peptide synthetases (NRPS) is located on RifA and recognizes AHBA as the starter unit (Admiraal et al., 2001
, 2002
). This is processively extended by malonyl-CoA and methylmalonyl-CoA via ten individual modules of a type I polyketide synthase (PKS) to give rise to the polyketide backbone of rifamycin. The amide synthase (RifF) catalyses the release of this linear product from the PKS complex and its cyclization via intramolecular amide formation. Inactivation of RifF results in the premature release of a series of polyketides of different chain length from the enzyme complex (Stratmann et al., 1999
; Yu et al., 1999
), and reveals that the formation of the naphthalene ring of rifamycin occurs during the chain extension. However, further studies are required to determine the mechanism and genes responsible for the construction of the naphthalene core motif.
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METHODS |
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Instrumentation.
Electrospray ionization mass spectrometry (ESI-MS) was carried out on a Bruker-Esquire ion trap mass spectrometer with electrospray, atmospheric pressure chemical ionization (APCI) or nanospray ionization sources, and high-resolution ESI-MS was carried out on a Fison VG Quattro II electrospray ionization mass spectrometer. Proton NMR spectra were recorded on a Bruker DRX-499 spectrometer with an SGI O2 computer. An ISF-4-V culture shaker (Adolf Kuhner AG) was used for the fermentation of A. mediterranei S699. Ultraviolet spectroscopy was carried out on a Hewlett Packard 8452A diode array spectrophotometer. For HPLC, a Beckman System Gold Programmable Solvent Module was used with a Beckman System Gold Diode Array Detector Module.
DNA manipulation.
Routine genetic procedures such as genomic and plasmid DNA isolations, restriction endonuclease digestions, alkaline phosphatase treatments, DNA ligations, and other DNA manipulations were performed according to standard techniques (Kieser et al., 2000; Sambrook et al., 1989
). DNA fragments were excised from agarose gels and residual agarose was removed with the QiaQuick Gel Extraction Kit (Qiagen). For Southern hybridizations, the genomic DNA was immobilized on a Hybond-N+ membrane (Amersham Pharmacia Biotech). Hybridization was performed at 68 °C for 5 h using nick-translated [32P]dCTP (PerkinElmer Life Sciences)-labelled DNA probes. Stringency washes were done with 0·1x SSC at 68 °C.
Plasmid DNA construction
Plasmids carrying disrupted or truncated genes used in the inactivation experiments were constructed as follows.
pTM0031AB.
A 5·6 kb FspI/SpeI fragment containing the region from part of orf16 to part of the rpoB gene, the very end of the rifamycin B biosynthetic gene cluster, and a 6·8 kb FspI/FspI fragment containing the region from part of rifN to part of orf5, were individually cloned from the cosmid clone pFKN108 into the pBluescript II SK() vector at SmaI/SpeI and EcoRV multiple cloning sites, respectively, providing plasmids pTM0031A and pTM0031B (Fig. 2). Combination of the blunt ends FspI with SmaI and FspI with EcoRV generated new recleavable sites AciI and SfaNI on pTM0031A and pTM0031B, respectively. Plasmid pTM0031B was then digested with KpnI and the resulting 4·5 kb KpnI/KpnI fragment was inserted into KpnI-digested pTM0031A to give pTM0031AB.
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Disruption of rif-orf5.
pTM4502 was digested with ApaI and then incubated with Klenow enzyme in the same way as rif-orf4 to give pTM45025
Disruption of rif-orf9.
A 7·1 kb BsiWI/BsiWI DNA fragment from pFKN108 (Fig. 3) containing part of rif-orf9, rif-orfs 10, 11, 17, 18 and part of rif-orf19 was blunt-ended with Klenow enzyme and subcloned into pBluescript II SK() at the EcoRV site to give pTM0035. Deletion of a 3·3 kb SphI/SphI fragment of pTM0035 provided pTM0035S, which was then digested with SmaI to give a 3·1 kb and a 3·7 kb fragment. The 3·7 kb fragment, containing the vector and part of rif-orf9, was recovered from agarose gel and religated to give pTM0035S9.
Disruption of rif-orf10.
pTM0035S was digested with SacI/NcoI to give a 6·4 kb and a 0·4 kb fragment. The 0·4 kb fragment (part of orf10) was recovered from the agarose gel, blunt-ended using Klenow enzyme and inserted into pBluescript II SK() at the EcoRV site to give pTM0035S10.
Disruption of rif-orfs 11 and 18.
pTM0035 was partially digested with MluI (0·1 unit µl1) to produce linear DNA with a cutting site within orf11, or within orf18. The cut plasmid DNA was blunt-ended using Klenow enzyme and dNTP mixture, and then religated. The obtained plasmids had either orf11 or orf18 disrupted and could be discriminated by digesting the plasmids with MluI and XbaI. The one with a disrupted orf11 was designated pTM00351 and that with a disrupted orf18, pTM00358
Disruption of rif-orf13.
A 3·4 kb StuI/XmnI DNA fragment from pFKN108 (Fig. 3) containing part of rif-orf20, rif-orfs 12, 13, 14 and part of rif-orf15 was cloned into pBluescript II SK() at the EcoRV site to give pTM1304. The plasmid was partially digested with NcoI (1 unit µl1) and the cut plasmid DNA was blunt-ended using Klenow enzyme and dNTP mixture, and then religated with T4 DNA ligase. The obtained plasmids had either orf12 or orf13 disrupted and could be discriminated by digesting the plasmids with NcoI/EcoNI or NcoI/XbaI. The desired construct was designated pTM1304K.
Disruption of rif-orf16.
A 3·6 kb NotI/ApaI DNA fragment from pFKN108 (Fig. 3), containing part of rif-orf15, rif-orf16 and rifJ, was cloned into pBluescript II SK() at the NotI/ApaI sites to give pTM1601. The plasmid was digested with BamHI and the cut plasmid DNA was blunt-ended using Klenow enzyme and dNTP mixture, and then religated to give pTM1601K.
Disruption of rif-orf19.
pTM1303 (Xu et al., 2003; Table 1
) was digested with HindIII, StuI and EcoRI to produce a 3·2 kb and a 5·9 kb fragment. The 3·2 kb fragment containing rif-orf19 was inserted into pBluescript II SK() at the HindIII/StuI site to give pTM1901. The 5·9 kb fragment containing rif-orf14 was inserted into pBluescript II SK() at the StuI/EcoRI site to give pTM1401 (Xu et al., 2003
). pTM1901 was then digested with BglII and incubated with Klenow enzyme as previously described to give pTM1901B.
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Construction of pTM1312.
pTM1303 was digested with MluI to produce a 5·7 kb fragment containing part of rif-orf14, rif-orfs 13, 12, 20, 19, and part of rif-orf18. The fragment was inserted into pAMR1 at the MluI site to give pTM1312.
Electrotransformation of the plasmid DNAs.
All plasmid DNAs were methylated with A. mediterranei S699 cell extract and S-adenosyl-L-methionine before use. Transformation of heat-denatured methylated plasmid DNAs into A. mediterranei S699 was carried out by electroporation at 12·5 kV cm1, and the transformants were immediately transferred to a 500 ml Erlenmeyer flask containing 50 ml YMG medium and incubated at 200 r.p.m. and 28 °C for 824 h. Single crossover mutants were selected by plating out the cells onto YMG plates containing hygromycin B, which were incubated at 30 °C for 714 days. The genotypes of mutant candidates were confirmed by Southern hybridization. Double crossover mutants were selected after three rounds of cultivation of a single crossover mutant in YMG liquid medium by replica plating on YMG agar plates with and without hygromycin B. Colonies that lost the antibiotic resistance were selected and genotypically verified by Southern hybridization as either mutants or revertants. The expected mutant strains were used for further analysis.
Isolation and structure elucidation of rifamycin intermediates.
Spores of the mutant strain were initially grown in YMG medium for 3 days at 28 °C and 200 r.p.m. This seed culture was used to inoculate (10 %, v/v) twenty 500 ml Erlenmeyer flasks containing 50 ml of YMG medium. After incubation for 10 days under the same conditions, the cultures were centrifuged, the pooled supernatants acidified to pH 3 with 1 M HCl, and the metabolites extracted with 3x100 ml ethyl acetate. Rifamycin-related compounds were purified consecutively by SiO2 gravity column chromatography (step gradient ethyl acetate to ethyl acetate/acetone 9 : 1, v/v) and HPLC (YMC.PAK ODS C18 10x250 mm, MeOH/water 78 : 22, v/v).
Rifamycin W: ESI-MS m/z: 654 (M-H). NMR: 1H (CD3OD, 499 MHz) (p.p.m.): 0·44 (3H, d, J=7 Hz, H-32), 0·72 (3H, d, J=7 Hz, H-33 or H-34), 0·92 (3H, d, J=7 Hz, H-31), 1·06 (3H, d, J=7 Hz, H-33 or H-34), 1·761·81 (2H, m, H-24 and H-26), 1·841·87 (1H, m, H-22), 1·95 (3H, s, H-14), 2·04 (3H, br s, H-13), 2·08 (3H, br s, H-30), 2·302·36 (1H, m, H-20), 2·592·64 (1H, m, H-28), 3·52 (1H, dd, J=2 and 10 Hz, H-25), 3·56 (1H, dd, J=7·5 and 11 Hz, H-34aa), 3·62 (1H, dd, J=6·5 and 11 Hz, H-34ab), 4·04 (1H, dd, J=1 and 10 Hz, H-23), 4·22 (1H, dd, J=1 and 10 Hz, H-21), 4·34 (1H, s, H-27)*, 6·11 (1H, dd, J=6·5 and 16 Hz, H-19), 6·25 (1H, d, J=11 Hz, H-17), 6·46 (1H, d, J=10 Hz, H-29), 6·54 (1H, dd, J=11 and 16 Hz, H-18), and 7·46 (1H, s, H-3). *J26,27 and J27,28=0 Hz (Nakata et al., 1990
).
Treatment of A. mediterranei S699 with ancymidol.
Ancymidol (12 mg) was added in two portions to cultures of wild-type strain S699 at 48 and 96 h after inoculation and the products were harvested after 710 days' cultivation. Extraction, purification and structure elucidation of the products were carried out according to the procedures described above.
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RESULTS |
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Inhibition of cytochrome P450 monooxygenases by ancymidol
To confirm the involvement of a cytochrome P450 monooxygenase in the conversion of rifamycin W, cultures of wild-type A. mediterranei S699 were treated with ancymidol, an inhibitor of cytochrome P450 monooxygenases (Ralston et al., 2001), and analysed for compound production. No apparent cell growth inhibition was detected in cultures with ancymidol, relative to those without ancymidol. However, cultures in the presence of ancymidol produced a dark brown colour, which turned to bright yellow when the pH of the culture was adjusted to 2·5 with 0·1 M H2SO4, whereas those without ancymidol have a light brown colour, indicating a normal production of rifamycin B. TLC, HPLC and ESI-MS analyses of the products revealed that in the presence of ancymidol the bacteria accumulated rifamycin W, supporting the role of a cytochrome P450, presumably Rif-Orf5, in the conversion of rifamycin W to DMDARSV.
Inactivation of rif-orf3
Based on results of the present study and others (Stratmann et al., 1999; Yu et al., 1999
), it was clear that the core naphthalene ring moiety of rifamycin is constructed during the polyketide chain extension process. However, the fact that the reaction may require a catalytic enzyme, whose gene is located in the post-PKS modification region and functions in trans with the PKS, is rather unusual. This notion has led us to a closer inspection of a functionally unknown gene (rif-orf3) that is located in the cluster directly upstream of the cytochrome P450-dependent monooxygenase genes rif-orfs 4 and 5. This gene originally showed no similarity to any known proteins in the database (but recently has been found to share high homology with a number of hypothetical proteins predicted from genome sequences; see Discussion). In silico analysis of the amino acid sequence revealed the presence in the protein of a putative flavin-nucleotide-binding region (Marchler-Bauer et al., 2003
), which might be involved in a phenolic hydroxylation reaction, making this gene an intriguing candidate for gene inactivation experiments. Therefore, disruption of rif-orf3 was implemented by an internal deletion of a 0·2 kb KpnI/KpnI fragment from the gene, and the mutant phenotype was analysed by HPLC and ESI-MS. The mutant gave an identical phenotypic pattern to that of the wild-type strain, but produced about 40 % more rifamycin B than the wild-type, suggesting that rif-orf3 is involved in the regulation of rifamycin B production rather than in the formation of the naphthalene ring.
Involvement of Rif-Orf19 in naphthalene ring formation
As neither the P450 monooxygenases nor rif-orf3 are directly involved in naphthalene ring formation, we then focused on rif-orf19, which encodes a protein homologous to 3-(3-hydroxyphenyl)propionate hydroxylases, a group of flavoproteins that catalyse the hydroxylation of phenol structures. This includes the 3-(3-hydroxyphenyl)propionic acid hydroxylases (HppA or MhpA) from Rhodococcus globerulus (32 % identity, 46 % similarity) (Barnes et al., 1997), E. coli strain K-12 (35 % identity, 51 % similarity) (Blattner et al., 1997
) and Comamonas testosteroni TA441 (37 % identity, 50 % similarity) (Arai et al., 1999
). Genes similar to rif-orf19 have also been reported to be involved in a number of secondary metabolite biosyntheses, such as in the biosyntheses of the antitumour antibiotic geldanamycin (gdmM; 46 % identity and 59 % similarity at the protein level) (Rascher et al., 2003
) and of the aromatic myxobacterial electron transport inhibitor stigmatellin (stiL; 27 % identity, 41 % similarity at the protein level) (Gaitatzis et al., 2002
). In silico analysis of the amino acid sequences revealed that all the proteins within this family contain typical nucleotide-binding motifs for flavoprotein hydroxylases with the amino acid sequences GxGxxG, DGxxSxxR and GDxxH (Fig. 5
) (Enroth et al., 1998
; Eppink et al., 1997
).
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Complementation of rif-orf19
To confirm the direct involvement of rif-orf19 in rifamycin B biosynthesis, a complementation experiment was carried out by introducing the wild-type rif-orf19 gene into the mutant strains using a replicating vector derived from plasmid pULVK2 (Kumar et al., 1994). Thus, a 5·7 kb MluI/MluI fragment of pTM1303 harbouring the complete operon of rif-orfs 13, 12, 20 and 19 was cloned into pAMR1, a pULVK2 (Kumar et al., 1996
) plasmid modified by the laboratory of Professor C. R. Hutchinson at the University of Wisconsin by introducing hygR and a multiple cloning site (MCS). The resulting plasmid (pTM1312) was electrotransformed into mutant strain MT1901BH and the transformants were selected on YMG plates containing the antibiotic hygromycin. Positive transformants could be easily identified by the diffusion of red-brown colour around the colonies on agar plates, which was further analysed by ESI-MS to show the recovery of rifamycin B production in these transformants. To confirm their genetic make-up, recovery of the plasmids was attempted by electroduction (Tuteja et al., 2000
) of the plasmid from the transformants into E. coli using procedures suggested by Lal and co-workers (Tuteja et al., 2000
); however, no plasmid could be recovered. On the other hand, Southern hybridization of the total cellular DNA using a 1·5 kb BglII/HindIII fragment of pTM1901 containing parts of rif-orf18 and rif-orf19 and a 1·7 kb fragment containing the hygR gene as probes gave strong signals, albeit in DNA of sizes that were different from those predicted for either the free plasmid or for plasmid that has been accidentally incorporated into the chromosome. This could be due to rearrangement and/or deletion of DNA fragments of pTM1312 in A. mediterranei, as such events have previously been reported for pULVK2 derivatives (Tuteja et al., 2000
). While the restriction patterns could not be readily explained, the strong hybridization signals for both probes indicated the presence of both rif-orf19 and hygR genes in the cells. No signal could be detected in the Southern hybridization of wild-type and mutant MT1901BH DNA using the hygR gene as a probe.
Inactivation of rif-orfs 9, 10, 11 and 18
Finally, to determine if any of the following genes rif-orf9 (putative aminotransferase), rif-orf10 (putative oxidoreductase), rif-orf11 (putative reductase) and rif-orf18 (putative 2,3-dehydratase) are involved in rifamycin B biosynthesis, they were individually inactivated by either single or double crossover recombination. Mutants generated by these experiments were grown in YMG medium and the products analysed by LC-MS. All mutants produced rifamycin B in amounts comparable to the wild-type, indicating that those gene products are not involved in rifamycin B biosynthesis.
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DISCUSSION |
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The aminodeoxyhexose genes (rif-orfs 610) in the cluster have been proposed to be functionally silent (August et al., 1998). This notion is based on the fact that no glycosylated rifamycins are found in cultures of A. mediterranei S699. On the other hand, glycosylated rifamycin congeners (tolypomycins) have been isolated from another rifamycin B producer, Streptomyces tolypophorus (Kishi et al., 1969
, 1972
). Previous work on these genes revealed that inactivation of rif-orfs 6 and 9, which encode proteins homologous to dNTP-hexose dehydratase (RfbH) of Salmonella enterica and aminotransferase (YokM) of Bacillus subtilis, respectively, results in mutants that produce rifamycin B, indicating that there is no direct involvement of these genes in rifamycin B biosynthesis (August et al., 1998
; M. Müller and others, unpublished results). These findings have been confirmed by the results of our experiments, as individual inactivation of rif-orfs 9, 10, 11, and 18 also does not affect rifamycin B production.
The first cyclization product released from the rif PKS is believed to be proansamycin X, a hypothetical molecule that has never been isolated or identified. It was expected that deletion of the entire set of post-PKS modification genes from the chromosome might give mutants that produce proansamycin X. However, mutant strain MT0031ABH, in which a 21 kb fragment of the post-PKS modification genes has been deleted, did not accumulate proansamycin X, but instead the tetraketides SY4b and desacetyl-SY4b were produced. These compounds have previously been isolated from knock-out mutants of the amide synthase RifF, as a result of premature release of the polyketide chain intermediates from the enzyme, starting from the tetraketide through the undecaketide (Stratmann et al., 1999; Yu et al., 1999
). Structure identification of these intermediates showed that from the pentaketide onwards they contain a naphthalene ring, suggesting that the formation of the naphthalene moiety of rifamycin B takes place during the chain extension of the polyketide, specifically between the tetraketide and the pentaketide stage. Therefore, the production of desacetyl-SY4b by MT0031ABH indicates that a gene(s) responsible for the formation of the naphthalene ring is located in the deleted region. However, attempts to complement the missing genes with a 28·1 kb EcoRV/SpeI fragment, harbouring all genes of the deleted region (Fig. 2
), have not been successful. It has been proposed that the formation of the naphthalene ring would require the introduction of an oxygen into the aromatic ring at the position para to the hydroxyl group of AHBA, followed by quinone formation and cyclization by a Michael addition (Floss & Yu, 1999
). The initial reaction might be catalysed by one of the cytochrome P450 monooxygenases (Fig. 6
). However, individual inactivation of rif-orfs 4, 13 and 16 did not significantly affect rifamycin B production. Interestingly, the rif-orf16 mutant produces both rifamycins SV and B, which raises a question whether rif-orf16 may to some extent be involved in the formation and/or attachment of the glycolate side chain.
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The hydroxylation of ACP-bound tetraketide by an FAD-dependent hydroxylase also raises an important question regarding the nature of substrate access to the active site. Mechanistically, this class of enzymes requires the co-factors FAD and NADPH, molecular oxygen, and substrate. FAD is in general incapable of catalysing the hydroxyl transfer reaction free in solution; therefore, the catalytic site must be shielded from solvent during the critical step of the hydroxylation reaction. As the substrate is rather bulky and is bound to a large complex protein (PKS), it is less likely that Rif-Orf19 adopts a regular open-closed channel mechanism for substrates and products to enter and leave the active site. The most attractive hypothesis would be that Rif-Orf19 docks onto the PKS and engulfs' the tetraketide portion of the ACP-bound substrate, whereas the PKS itself serves as a giant lid to the active site of the hydroxylase. If this is the case, how and when NADPH and molecular oxygen enter the active site would be open questions. Interestingly, alignment analysis of the amino acid residues of Rif-Orf19 with those of related enzymes showed no significant disparity at the sequence levels (Fig. 5). Instead, all proteins seem to be highly similar, particularly Rif-Orf19 and GdmM (46 % identity, 59 % similarity); the latter is involved in geldamycin biosynthesis (Rascher et al., 2003
). However, there are a number of amino acid residues in Rif-Orf19 which are different from the conserved residues found in other hydroxylases (indicated by large arrows in Fig. 5
) and which may play a role in its potentially distinct structural and/or catalytic nature. Detailed biochemical and structural investigations of the protein are critical to address these intriguing questions.
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ACKNOWLEDGEMENTS |
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Received 20 April 2005;
accepted 16 May 2005.
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