J Biol Chem, Vol. 275, Issue 5, 3065-3074, February 4, 2000

Characterization of Two Polyketide Methyltransferases Involved in the Biosynthesis of the Antitumor Drug Mithramycin by Streptomyces argillaceus^*

M. José Fernández Lozano^§, Lily L. Remsing^¶, Luis M. Quirós, Alfredo F. Braña, Ernestina Fernández, César Sánchez, Carmen Méndez, Jürgen Rohr^¶, and José A. Salas^**

From the  Departamento de Biología Funcional e Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain and the ^¶ Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425-2303

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A DNA chromosomal region of Streptomyces argillaceus ATCC 12596, the producer organism of the antitumor polyketide drug mithramycin, was cloned. Sequence analysis of this DNA region, located between four mithramycin glycosyltransferase genes, showed the presence of two genes (mtmMI and mtmMII) whose deduced products resembled S-adenosylmethionine-dependent methyltransferases. By independent insertional inactivation of both genes nonproducing mutants were generated that accumulated different mithramycin biosynthetic intermediates. The M3MI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3M2 (mtmMII-minus mutant) accumulated 9-demethylpremithramycin A3 (9-DPMA3), premithramycin A1 (PMA1), and 7-demethylmithramycin, all of them containing the O-methyl group at C-4 and C-1', respectively, but lacking the methyl group at the aromatic position. Both genes were expressed in Streptomyces lividans TK21 under the control of the erythromycin resistance promoter (ermEp) of Saccharopolyspora erythraea. Cell-free extracts of these clones were precipitated with ammonium sulfate (90% saturation) and assayed for methylation activity using different mithramycin intermediates as substrates. Extracts of strains MJM1 (expressing the mtmMI gene) and MJM2 (expressing the mtmMII gene) catalyzed efficient transfer of tritium from [³H]S-adenosylmethionine into 4-DPMC and 9-DPMA3, respectively, being unable to methylate other intermediates at a detectable level. These results demonstrate that the mtmMI and mtmMII genes code for two S-adenosylmethionine-dependent methyltransferases responsible for the 4-O-methylation and 9-C-methylation steps of the biosynthetic precursors 4-DPMC and 9-DPMA3, respectively, of the antitumor drug mithramycin. A pathway is proposed for the last steps in the biosynthesis of mithramycin involving these methylation events.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Actinomycetes, and particularly streptomycetes, are producers of a great variety of bioactive compounds, many of which have clinical application as antibiotics, antifungal, antiparasites, or antitumor drugs. Many of these bioactive drugs contain methyl groups in their structures, which are introduced during biosynthesis. However, not all these methylations are due to the action of methyltransferases. In the case of the polyketides, some of these methyl groups derive from the carboxylic acids used as starter or extender units during the condensation reactions (1). In other cases, the action of specific methyltransferases is responsible for the methylation reactions. Most of them are O-methylations, as is the case of tetracenomycin C (2, 3), daunorubicin (4-6), erythromycin (7), and tylosin (8). But also N-methylations (9) and C-methylations (10, 11) have been described. As far as they have been characterized, most of the methyltransferases participating in antibiotic biosynthesis use S-adenosylmethionine as cofactor, although biotin and tetrahydrofolate can be alternatively used by other methyltransferases.

Mithramycin (also designated as aureolic acid, plicamycin, mithracin, LA-7017, PA-144; Fig. 1A) is an aromatic polyketide which shows antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria because a permeability barrier. It also shows a remarkable cytotoxicity against a variety of tumor cell culture lines, including brain tumors and experimental animal tumors. Mithramycin has been clinically used for the treatment of certain tumors, such as disseminated embryonal cell carcinoma as well as for Paget's bone disease (12, 13) and also finds use for control of hypercalcemia in patients with malignant disease (14). Mithramycin, together with the chromomycins, olivomycins, chromocyclomycin, and UCH9, constitutes the aureolic acid group of antibiotics. All they belong to the large and important family of the aromatic polyketides. Great progress has been made in the last few years on the understanding of the mithramycin biosynthetic pathway. It has been shown that the biosynthesis of the mithramycin aglycon derives from the condensation of 10 acetate units in a series of reactions catalyzed by a type II polyketide synthase thus generating a decaketide (15). After several aromatizations and cyclizations two nonglycosylated intermediates are synthesized, 4-demethylpremithramycinone (4-DPMC)¹ and premithramycinone (PMC) (16, 17). This last compound is later glycosylated with (in this order) a trisaccharide (D-olivose-D-oliose and D-mycarose) and a disaccharide (D-olivose-D-olivose) (18, 19). Finally, as one of the last events in mithramycin biosynthesis, an oxidative breakage (followed by a keto reduction) of the fourth ring of the fully glycosylated intermediate premithramycin B takes place generating the final mithramycin molecule (20). The structure of mithramycin bears nine methyl groups. C-5', which is derived from the acetate starter unit, the O-methyl group at C-1'-O and the methyl side chain at C-7 of the polyketide-derived aglycon moiety. Furthermore, five methyl groups (always C-6 of each deoxysugar unit) are derived from the dehydration step affecting C-4 and C-6 of the corresponding sugars during an early step in the deoxysugar biosynthesis). Finally, another methyl side chain is attached at C-3 of the D-mycarose, the third sugar of the trisaccharide chain. Here we report the cloning, sequencing, and characterization of two genes (mtmMI and mtmMII) encoding two S-adenosylmethionine-dependent methyltransferases. Insertional inactivation of both genes and in vitro methylation assays demonstrated that the MtmMI and MtmMII methyltransferases are responsible for introduction of methyl groups at C-1'-O and C-7, respectively, of the polyketide moiety of mithramycin by Streptomyces argillaceus. These studies have allowed the identification of the possible substrates for the two methyltransferases and to propose a pathway for the methylation steps in mithramycin biosynthesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Microorganisms, Culture Conditions, and Vectors

Streptomyces argillaceus ATCC 12596, mithramycin producer, was used as donor of chromosomal DNA. For sporulation on solid medium, it was grown at 30 °C on plates containing A medium (21). For growth in liquid medium, the organisms were grown either on TSB medium (trypticase soya broth, Oxoid) or R5A medium (18). Streptomyces lividans TK21 was used as host for gene expression. When plasmid-containing clones were grown, the medium was supplemented with the appropriate antibiotics: 5 or 50 µg/ml thiostrepton for liquid or solid cultures, respectively, 100 µg/ml for ampicillin, 25 µg/ml apramycin, or 20 µg/ml tobramycin. Escherichia coli XL1-Blue (22) and E. coli XL2-Blue (Stratagene) were used as hosts for subcloning. pUC18 was used for sequencing and subcloning. pIAGO² is pWHM3 (24) containing the promoter of the erythromycin resistance gene (ermE) from Saccharopolyspora erythraea as a 0.28-kb KpnI-BamHI fragment. pBSKT is a pBluescript derivative containing a thiostrepton resistance cassette (25). pEFBA is a pBluescript derivative containing an apramycin resistance cassette.

DNA Manipulation

Plasmid DNA preparations, restriction endonuclease digestions, alkaline phosphatase treatments, DNA ligations, and other DNA manipulations were performed according to standard techniques for E. coli (26) and Streptomyces (27). Preparation of Streptomyces protoplasts, transformation, and selection of transformants was carried out as in described in Ref. 27 with minor modifications for S. argillaceus (20).

DNA Sequencing

Sequencing was performed on double-stranded DNA templates by using the dideoxynucleotide chain termination method (28) and the Cy5 AutoCycle Sequencing Kit (Amersham Pharmacia Biotech). Both DNA strands were sequenced with primers supplied in the kits or with internal oligoprimers (17-mer) using an ALF-express automatic DNA sequencer (Amersham Pharmacia Biotech). Computer-aided data base searching and sequence analyses were carried out using the University of Wisconsin Genetics Computer Group programs package (UWGCG; Ref. 29) and the BLAST program (30).

Insertional Inactivation

The mtmMI and mtmMII genes were inactivated by gene replacement as follows.

mtmMI-- A 3.4-kb BamHI-SphI fragment (sites 6-13 in Fig. 1B) was subcloned into the same sites of pUC18. The internal 0.5-kb PstI fragment (sites 10 to 11 in Fig. 1B) to mtmMI was then deleted by PstI digestion and replaced by a 1.5-kb PstI fragment from pEFBA containing an apramycin resistance cassette. The insert in this construct was rescued by EcoRI-HindIII digestion (these restriction sites from the pUC polylinker) and subcloned into the sames of pBSKT, generating pMFMI-1. This construct was used to transform S. argillaceus protoplasts and one apramycin-resistant transformant was isolated being also thiostrepton-resistant and therefore the consequence of a single crossover. Spores from this clone were used to inoculate TSB liquid medium without any antibiotic and, after incubation at 30 °C for 72 h, protoplasts were prepared, diluted onto P medium (27), and plated onto R5 agar plates containing apramycin (25 µg/ml). Surviving colonies were tested for thiostrepton sensitivity and two colonies were found to be thiostrepton-sensitive as a result of a second crossover. One of them, M3MI, was chosen fo further studies.

mtmMII-- A 2.2-kb BamHI-PstI fragment (sites 6 to 10 in Fig. 1B) was subcloned into the same sites of pUC18. Then, an internal 0.3-kb SalI fragment (sites 7 to 8 in Fig. 1B) in mtmMII was deleted by SalI digestion, followed by end filling with Klenow polymerase and then replaced by a 1.5-kb SmaI-EcoRV fragment containing the apramycin resistance cassette. Finally and to incorporate a second antibiotic resistance marker into the vector, a 1.4-kb SmaI fragment containing the thiostrepton resistance gene was subcloned into the unique ScaI site of pUC18 located outside the polylinker. This construct (pMFMII-1) was used to transform S. argillaceus protoplasts and two apramycin-resistant transformants were isolated being also thiostrepton-resistant and therefore the consequence of a single crossover. The occurrence of a second crossover was forced by cultivating one of these clones in TSB medium without antibiotic, as in the case of the M3MI mutant (see above). One apramycin-resistant thiostrepton-sensitive colony, M3MII, was isolated and further studied.

Gene Expression

For expression in S. lividans of both mtmMII and mtmMI genes, a 3.4-kb BamHI-SphI fragment (sites 6 to 13 in Fig. 1B) containing both genes was subcloned into the same restriction sites of pIAG0 generating plasmid pEFM45. For expression of mtmMII gene, pEFM45 was digested with PstI and then religated generating pMJM2. In this construction, the mtmMI gene was inactivated by deleting of the internal 0.5-kb PstI fragment. For expression of mtmMI gene, the gene was amplified by PCR using the following primers: 5'-CGCGGATCCTCGATCGAAAGGCACGCGATG-3' for the 5'-end of the gene and 5'-CCCAAGCTTTCAGCCGGGCTTGCGGG-3' for the 3'-end of the gene (BamHI and HindIII sites, respectively, were included in the oligoprimers to facilitate subcloning and are indicated in bold). The PCR product was subcloned into the BamHI-HindIII sites of pIAGO, generating pMJM1. The two constructs (pMJM1 and pMJM2) were introduced into S. lividans TK21 by protoplast transformation and transformants selected for thiostrepton resistance (50 µg/ml).

PCR Amplification

PCR reaction conditions were as follows: 100 ng of template DNA were mixed with 30 pmol of each primer and 2 units of Vent DNA Polymerase (New England Biolabs) in a total reaction volume of 50 µl containing 2 mM of each dNTP, 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄, and 0.1% Triton X-100. This reaction mixture was overlaid with 50 µl of mineral oil (Sigma). The polymerization reaction was performed in a thermocycler (MinyCycler, MJ Research) under the following conditions: an initial denaturation of 3 min at 98 °C; 30 cycles of 30 s at 98 °C, 1 min at 61.6 °C, and 1.2 min at 72 °C; after the 30 cycles, an extra extension step of 5 min at 72 °C was added.

Preparation of Cell-free Extracts

50 ml of TSB medium were inoculated with a spore suspension of the different S. lividans clones in the presence of 5 µg/ml thiostrepton. After 2 days of incubation at 30 °C, the mycelia was collected by centrifugation and washed twice with 50 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol and 1 mM EDTA. The mycelia was suspended in a small volume of the same buffer and disrupted by sonication (10 pulses 20 s each with intermittent cooling in a MSE-ultrasonic disintegrator at 150 W and 20 kHz). After removal of unbroken cells and cellular debris by centrifugation, the soluble fraction was precipitated with ammonium sulfate (90% saturation) and after resuspension in a small volume of the buffer mentioned above extensively dialyzed against the same buffer.

Methylation Assays

Methylation reactions consisted of the following components (500 µl final volume): 10 µl of S-adenosyl-L-[methyl-³H]methionine (1 µCi/ml; specific activity 68 Ci/mmol), 10 µl of the correspondent mithramycin intermediate (0.2 µM final concentration) and a variable volume of extract and buffer (total 500 µl) depending on the protein concentration of the extracts. After incubation at 37 °C for 1 h, the reactions were stopped by adding one-tenth of their volume 0.2 N HCl and extracted twice with 500 µl of ethyl acetate. After phase separation, the organic phase was recovered, the solvent evaporated and the residue was suspended in 50 µl of methanol and the samples analyzed by HPLC. Fractions were collected and, after addition of scintillation mixture, the radioactivity in the samples was determined.

HPLC Analysis

Detection of mithramycin-related compounds was performed in a reversed phase column (Symmetry C18, 4.6 × 250 mm, Waters), with acetonitrile and 0.1% trifluoroacetic acid in water as solvents. A linear gradient from 10 to 100% acetonitrile in 30 min, at a flow rate of 1 ml/min, was used. Detection and spectral characterization of peaks were made with a photodiode array detector and Millennium software (Waters), extracting bidimensional chromatograms at 280 nm.

Isolation of Mithramycin Intermediates

Spores of mutant M3MII were initially grown in TSB medium during 24 h at 30 °C and 250 rpm. This seed culture was used to inoculate (at 2.5%, v/v) eight 2-liter Erlenmeyer flasks containing 400 ml of R5A medium. After incubation for 4 days in the above conditions, the cultures were centrifuged, filtered, and extracted as described (18). Mithramycin-related compounds (identified according to their spectral characteristics) were purified by preparative HPLC in a µBondapak C18 radial compression cartridge (PrepPak Cartridge, 25 × 100 mm, Waters). Short gradients using 0.1% trifluoroacetic acid in water and either methanol or acetonitrile, at 10 ml/min, were optimized for resolution of individual peaks. The purified material collected in each case was diluted 4-fold with water, applied to a solid-phase extraction column (Lichrolut RP-18, Merck), washed with water to eliminate trifluoroacetic acid, and finally eluted with methanol and dried in vacuo.

Physicochemical Data of 7-Demethylmithramycin and 9-Demethylpremithramycin A3

7-Demethylmithramycin-- ¹H NMR (400 MHz, acetone-d₆)  1.19 (s, 3C-Me), 1.20 (d, 6E-H₃, J = 6.0 Hz), 1.22 (d, 5'-H, J = 6.5), 1.24-1.31 (4 d, J = 6, 6A-H₃, 6B-H₃, 6C-H₃, 6D-H₃), 1.54 (ddd, 2B-H_a, J = 12, 12, 10), 1.54 (dd, 2E-H_a, J = 13.5, 9.5), 1.60 (ddd, 2C-H_a, J = 12, 12, 10), 1.73 (ddd, 2D-H_a, J = 12, 12, 10), 1.78 (ddd, 2A-H_a, J = 12, 12, 10), 1.88 (dd, 2E-H_e, J = 13.5, 2), 1.93 (ddd, 2D-H_e, J = 12, 4.5, 2), 2.16 (ddd, 2B-H_e, J = 12, 5, 2), 2.38 (ddd, 2A-H_e, J = 12, 5, 2), 2.54 (ddd, 2C-H_e, J = 12, 5, 2), 2.64 (dd, 4-H_e, J = 16, 3), 2.82 (bt, 3-H, J = 12), 2.9-2.95 (complex, 4-H_a), 2.90-3.04 (overlapping signals, 4A-H, 4B-H, 4C-H, 4E-H), 3.30 (dq, 5C-H, J = 9, 6), 3.36 (dq, 5B-H, J = 9, 6), 3.43 (s, 1'-OCH₃), 3.48-3.66 (overlapping signals, 3A-H, 3B-H, 3C-H), 3.54 (dq, 5A-H, J = 9, 6), 3.63 (dq, 5E-H, J = 9, 6), 3.71 (m, 5D-H), 3.71 (bs, 4D-H), 3.88 (ddd, 3D-H, J = 12, 5, 3), 4.23 (m, 3'-H, 4'-H), 4.70 (dd, 1D-H, J = 10, 2), 4.71 (dd, 1B-H, J = 10, 2), 4.78 (d, 2-H, J = 11.5), 4.83 (d, 1'-H, J = 1.5), 4.95 (dd, 1E-H, J = 9.5, 2), 5.06 (dd, 1C-H, J = 10, 2), 5.35 (dd, 1A-H, J = 10, 2), 6.44 (d, 7-H, J = 2), 6.76 (d, 5-H, J = 2), 6.87 (bs, 10-H) ppm.

¹³C NMR (100.6 MHz, acetone-d₆)  17.0 (C-6D), 18.0 (C-6B), 18.4 (C-6C), 18.5 (C-6A), 18.7 (C-6E), 20.1 (C-5'), 27.5 (3E-CH₃), 27.8 (C-4), 32.9 (C-2D), 37.9 (C-2A), 38.4 (C-2C), 40.4 (C-2B), 43.1 (C-3), 44.8 (C-2E), 59.1 (1'-OCH₃), 68.9 (C-4'), 69.3 (C-4D), 71.1 (C-3E), 71.4 (C-5D, C-5E), 71.8 (C-3B), 73.0 (C-5A, C-5C), 73.1 (C-5B), 75.8 (C-4A), 76.1 (C-4C), 77.2 (C-3D, C-2), 77.3 (C-4E), 78.0 (C-4B), 79.6 (C-3'), 81.7 (C-3A), 82.3 (C-3C, C-1'), 97.1 (C-1A), 98.4 (C-1E), 100.4 (C-1B), 100.7 (C-1D), 101.3 (C-1C), 102.3 (C-5), 103.4 (C-7), 108.7 (C-8a), 109.1 (C-9a), 117.6 (C-10), 138.2 (C-4a), 141.8 (C-10a), 160.2 (C-8), 161.7 (C-6), 165.9 (C-9), 204.6 (C-1), 211.7 (C-2') ppm.

FAB MS m/z (relative intensity) [M + Na]⁺ 1093.7 (16), [M + H]⁺ 1071.5 (3), [M  H] 1069.4 (48); HRFAB MS 1093.4364 (C₅₁H₇₄O₂₄ + Na, calculated 1093.4468).

9-Demethylpremithramycin-A3-- ¹H NMR (400 MHz, Acetone-d₆)  1.20 (s, 3C-Me), 1.20 (d, 6C-H₃, J = 6.1 Hz), 1.24 (d, 6A-H₃, J = 6.1 Hz), 1.26 (d, 6B-H₃, J = 6.5), 1.52 (dd, 2C-H_a, J = 13.5, 9.5), 1.55 (ddd, 2A-H_a, J = 12, 12, 9.5), 1.74 (ddd, 2B-H_a, J = 12, 12, 10), 1.86 (dd, 2C-H_e, J = 13.5, 2), 1.90 (ddd, 2B-H_e, J = 12, 4.5, 2), 2.57 (m, 2A-H_e, broad), 2.61 (s, 2'-H₃), 2.90 (dd, 4A-H, J = 9, 8.5), 2.91 (d, 4C-H, J = 9.5), 3.05 (dd, 5-H_e, J = 16.5, 3), 3.13 (ddd, 4a-H, J = 11, 4, 3), 3.29 (dq, 5A-H, J = 9, 6), 3.52 (s, 4-OMe), 3.58 (ddd, 3A-H, J = 12, 8.5, 5), 3.62 (dq, 5C-H, J = 9.5, 6), 3.65 (q, 5B-H, J = 6.5), 3.68 (s, 4B-H), 3.81 (m, 5-H_a, broad), 3.86 (ddd, 3B-H, J = 12, 4.5, 3), 4.21 (d, 4-H, J = 11), 4.63 (dd, 1B-H, J = 10, 2), 4.87 (dd, 1A-H, J = 9.5, 2), 4.93 (dd, 1C-H, J = 9.5, 2), 6.41 (d, 9-H, J = 2), 6.64 (d, 7-H, J = 2), 6.97 (bs, 6-H) ppm.

¹³C NMR (100.6 MHz, acetone-d₆)  16.9 (C-6B), 18.4 (C-6A), 18.7 (C-6C), 26.9 (C-5), 27.5 (3C-CH₃), 28.5 (C-2'), 32.9 (C-2B), 38.6 (C-2A), 43.0 (C-4a), 44.8 (C-2C), 61.8 (4-OCH₃), 69.3 (C-4B), 71.1 (C-3C), 71.4 (C-5B, C-5C), 72.9 (C-5A), 75.7 (C-4A), 77.2 (C-3B, C-4C), 77.9 (C-4), 82.2 (C-3A), 85.9 (C-12a), 98.3 (C-1A, C-1C), 100.7 (C-1B), 102.4 (C-9), 103.5 (C-7), 107.5 (C-10a), 108.4 (C-11a), 113.0 (C-2), 118.2 (C-6), 136.2 (C-5a), 143.0 (C-6a), 161.0 (C-10), 163.5 (C-8), 168.3 (C-11), 188.8 (C-3), 193.8 (C-12), 196.8 (C-1), 204.6 (C-1') ppm.

FAB MS m/z (relative intensity) [M + Na]⁺ 841 (100), [M + H]⁺ 819 (4), [M  H] 817 (51); HRFAB MS 841.2908 (C₄₀H₅₀O₁₈ + Na, calculated 841.2895).

Protein Determination

Protein estimation was carried out by measurement of absorbance at 540 nm by a protein-dye binding assay using bovine serum albumin as standard (31).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Sequencing of the mtmMI and mtmMII Genes-- Previous research in our laboratory on the mithramycin biosynthetic gene cluster allowed the isolation and characterization of a series of overlapping cosmid clones containing the mithramycin gene cluster. Through sequencing analysis in one of these cosmids, clone cosAR3, genes encoding two glycosyltransferases (18), an oxygenase (20), and an ABC transporter system (21) were identified. A region of approximately 2.2 kb located immediately upstream of the two glycosyltransferase genes mtmGII and mtmGI (Fig. 1B) has been now characterized. We determined the nucleotide sequence of the DNA region (Fig. 2) and analyzed for the presence of potential coding regions using the CODONPREFERENCE program (29) and two open reading frames (ORFs) transcribed in the same direction were found. The first ORF (designated mtmMII) starts in a ATG codon, comprises 978 nucleotides and ends in a TGA stop codon; it would code for a polypeptide of 329 amino acids and an estimated M_r of 35,529. The starting codon (ATG) of the second ORF (designated mtmMI) is separated 62 base pairs from the stop codon of mtmMII. It comprises 1,035 nucleotides and ends in a TGA codon; it would code for a polypeptide of 345 amino acids with an estimated M_r of 37,863. Both genes show a high GC content characteristic of Streptomyces genes and the bias in the third codon position characteristic of the genes of this genus. Downstream of mtmMI there is a DNA region with two long inverted repeats that could form stem-loop structures that might represent transcriptional terminators.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   A, Structure of mithramycin. The two methyl groups introduced by specific methyltransferases in the polyketide moiety of mithramycin are indicated by the arrows. B, schematic representation of the sequenced region of cosAR3 and location of the mtmMI and mtmMII genes with respect to the previously characterized genes from the mithramycin pathway. B, BamHI; S, SalI; P, PstI; H, SphI; N, NcoI.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of the 2259-base pair sequenced. The nucleotide sequence is shown with the deduced amino acid sequence of the different ORFs in single-letter code. Inverted repeated sequences are underlined with arrows. The sequence has been deposited in GenBank under accession number AF077869.

Deduced Functions of the MtmMI and MtmMII Proteins-- The deduced products of the mtmMI and mtmMII genes were compared with other proteins in data bases using the BLAST program (30). Both proteins showed similarities with methyltransferases from different sources. The MtmMI protein showed the highest similarity with a methylase (ORF3) possibly involved in tetracycline biosynthesis in S. aureofaciens, 47.2% identity (32), the TcmO O-methyltransferase from S. glaucescens involved in the biosynthesis of tetracenomycin C, 42.2% identity (2), the DnrK carminomycin 4-O-methyltransferase from S. peucetius involved in the conversion of carminomycin into daunorubicin, 26.4% identity (6), the DauK methyltransferase involved in daunomycin biosynthesis in Streptomyces sp. C5, 25.8% identity (33) and the DmpM O-demethylpuromycin O-methyltransferase from S. alboniger involved in puromycin biosynthesis, 26.3% identity (34). In addition it showed significant similarity to several O-methyltransferases involved in hydroxyindol and catechol methylation. The MtmMII protein showed the highest similarities with the RdmB methyltransferase from the rhodomycin pathway in S. purpurascens, 26.9% identity (35), the TcmN multifunctional cyclase-dehydratase-3-O-methyltransferase involved in methylation in the biosynthesis of tetracenomycin C by S. glaucescens, 25.8% identity (2), the TcmO O-methyltransferase, 25.1% identity (2), the DnrK carminomycin 4-O-methyltransferase, 25.3% identity (6) and the DauK methyltransferase, 25% identity (33). MtmMII also resembled catechol and hydroxyindol O-methyltransferases but the BLAST scores were smaller than those for the MtmMI BLAST searches. All these methyltransferases show three conserved motifs (Fig. 3) that are supposed to be involved in the binding of the S-adenosylmethionine cofactor and/or one of the products of the methylation reaction (S-adenosylhomocysteine) (36). Motif I, a glycine-rich region, is the most conserved of these three motifs in the different methyltransferases. It is usually separated from motif II by approximately 36-90 amino acid residues. Motif III is normally located between 12 and 38 amino acids from the end of motif II. In some methyltransferases not all these motifs are clearly identified. In the MtmMI and MtmMII proteins, the three motifs are clearly recognized (Fig. 3). On the basis of the profiles deduced from these data base comparisons it was assumed that mtmMI and mtmMII could code S-adenosylmethionine-dependent methyltransferases.

View larger version (118K): [in this window] [in a new window]   Fig. 3.   Alignment of the deduced amino acid sequences of the MtmMI and MtmII proteins with different methyltransferases. DauK, daunomycin biosynthesis in Streptomyces sp. C5 (33); DnrK, daunorubicin biosynthesis in S. peucetius (6); RdmB, rhodomycin biosynthesis in S. purpurascens (35); DmpM, puromycin biosynthesis in S. alboniger (34); TcmN, tetracenomycin biosynthesis in S. glaucescens (2); Orf3aureo, tetracycline biosynthesis in S. aureofaciens (32); TcmO, tetracenomycin biosynthesis in S. glaucescens (2); MtmMI and MtmMII, this paper.

Inactivation of the mtmMI and mtmMII Genes-- To assign a role to MtmMI and MtmMII in the mithramycin biosynthesis, both corresponding genes were independently inactivated, by deleting an internal DNA fragment and its replacement by an apramycin resistance cassette. In the case of the mtmMI gene, one of two apramycin-resistant thiostrepton-sensitive colonies obtained was selected for further characterization (M3MI mutant). Southern hybridization showed that the wild type region in the chromosome of this mutant was replaced by the in vitro mutated one (Fig. 4, A and B). Using as probe a 1.2-kb NcoI-StuI fragment (sites 9-12 in Fig. 1B), two BamHI hybridizing bands (3.6 and 2.8 kb) were observed in the M3MI mutant in comparison to the unique 5.3-kb BamHI band of the wild type strain (Fig. 4B). HPLC analysis of cultures of the M3MI mutant showed that it did not produce mithramycin, but a new HPLC peak was detected (Fig. 4C) with exactly the same HPLC mobility and absorption spectrum as a previously isolated mithramycin intermediate, 4-DPMC (Fig. 4C). This is the C-4-O non-methylated analogon of PMC and therefore its accumulation in the M3MI mutant suggests that the MtmMI methyltransferase converts 4-DPM into PMC. In the case of the mtmMII gene, one clone (mutant M3MII) was also selected resulting from a double crossover. Using as probe the 5.3-kb BamHI fragment (sites 6-14 in Fig. 1B), two hybridizing BamHI bands (2.5 and 3.9 kb) were observed in the M3MII mutant in comparison to the 5.3-kb BamHI band of the wild type strain (Fig. 5B). HPLC analysis of cultures of this mutant showed the presence of three major peaks. Peak b corresponded to another previously isolated mithramycin intermediate, PMA1 (18). To further characterize the compounds present in peaks a and c, they were purified by preparative HPLC and their structures elucidated.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Insertional inactivation of the mtmMI gene. A, scheme representing the replacement in the chromosome of the wild type mtmMI gene by the in vitro mutated one. B, BamHI; P, PstI; H, SphI; aac, apramycin resistance gene. The thin lines represent the BamHI fragments that hybridize with the probe. B, Southern hybridization using the 1.2-kb NcoI-StuI fragment as the 32P-labeled probe. Lane 1, BamHI-digested chromosomal DNA from the wild type strain. Lane 2, BamHI-digested chromosomal DNA from mutant M3MI. C, HPLC analysis of a culture of mutant M3MI. Peak d corresponds to 4-DPMC.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Insertional inactivation of the mtmMII gene. A, scheme representing the replacement in the chromosome of the wild type mtmMII gene by the in vitro mutated one. B, BamHI; EV, EcoRV; M, SmaI; P, PstI; S, SalI; aac, apramycin resistance gene. The thin lines represent the BamHI fragments that hybridize with the probe. B, Southern hybridization using the 5.3-kb BamHI fragment as the 32P-labeled probe. Lane 1, BamHI-digested chromosomal DNA from the wild type strain. Lane 2, BamHI-digested chromosomal DNA from mutant M3MII. C, HPLC analysis of a culture of mutant M3MII. Peak a corresponds to PMA1 and peaks a and c were selected for further study.

Structural Elucidation of the Accumulated Compounds-- The negative ion FAB MS spectra of the two new compounds support the following molecular formulae: m/z 1069.4, C₅₁H₇₄O₂₄ for 7-demethylmithramycin (peak a), and m/z 817, C₄₀H₅₀O₁₈ for 9-demethylpremithramycin A3 (peak c).

The former compound was elucidated to be 7-demethylmithramycin by comparison of the ¹H and ¹³C NMR data with that of mithramycin. In the ¹H NMR spectrum, the 7-CH₃ singlet at 2.11 ppm is missing, and an aromatic proton at 6.44 ppm can be observed instead. The ¹³C NMR spectrum supports the presence of a proton at the 7-position by the loss of the 7-CH₃ signal at 7.0 ppm and a shift in the 7-C signal from the characteristic quarternary carbon at 110.0 ppm to a dublet at  103.4 ppm (comparable to premithramycin A1). All other ¹H and ¹³C signals appeared to be like mithramycin. The ¹H and ¹³C NMR spectra for the latter compound were similar to that of premithramycin A3. All premithramycin A3 signals were present, except the 9-CH₃ signals at 2.16 and 8.2 ppm (¹H and ¹³C, respectively). Instead, an aromatic proton signal was observed at  6.41 and the 9-C signal was shifted from  110.7 (quarternary carbon) to 102.4 (doublet). Based on these observations, the compound was identified as 9-demethylpremithramycin A3.

Expression of the mtmMI and mtmMII Genes and Assays of Methylation-- In order to determine the putative substrates of the MtmMI and MtmMII methyltransferases, we decided to establish an in vitro methylation assay to test the activity of both methyltransferases. Both genes were independently expressed in S. lividans TK21. Plasmids pMJM1 and pMJM2 expressing the mtmMI or mtmMII gene, respectively, were introduced into S. lividans TK21 by protoplast transformation. One transformant (thiostrepton-resistant) was selected from each transformation (strains MJM1 and MJM2, respectively) and, after cultivation in TSB medium for 3 days at 30 °C, methylating activity against different mithramycin intermediates was tested using extracts obtained as described under "Experimental Procedures." As potential substrates for methylation several mithramycin intermediates were tested (Fig. 7): 4-DPMC (17, 20) and PMC (17, 37), premithramycin A1 (PMA1) (18), 9-demethylpremithramycin A3 (9-DPMA3; isolated in this work) and 7-demethylmithramycin (7-DMTM) (isolated in this work). 4-DPMC is a tetracyclic nonglycosylated intermediate that lacks both, the O-methyl group at C-4 and the methyl side chain at C-9 (20). PMC is the C-4 methylated derivative of 4-DPMC. PMA1 is a tetracyclic compound containing a D-olivose sugar unit attached to C-12a-O and lacking the C-methyl group at C-9. For TLC and HPLC comparison, we used premithramycin A2 (PMA2) and premithramycin A3 (PMA3) (18) as potential products of the methylation reactions. PMA2 is similar to PMA1, but contains a disaccharide (D-olivose-D-oliose) and possesses already the C-methyl group at C-9. The latter is also true for PMA3, which has a structure like PMA2 plus a D-mycarose unit attached to the disaccharide D-olivose-D-oliose of the latter. Extracts of strain MJM1 were very active in the transfer of tritium from [³H]S-adenosylmethionine into 4-DPMC (Fig. 6A) but not to PMC and the other substrates tested (data not shown). Tritium transfer was dependent on the presence of 4-DPMC since no transfer of radioactivity was detected in the absence of 4-DPMC (Fig. 6A). The products of the reaction were analyzed by HPLC (Fig. 6B) and radioactivity of the different peaks counted (Fig. 6C). Most of the radioactivity was associated with a fraction, which corresponded to PMC (Fig. 6C), indicating that the MtmMI methyltransferase converts 4-DPMC into PMC. When using extracts of the MJM2 strain only 9-DPMA3 was efficiently methylated (Fig. 7A). Again HPLC analysis of the reaction products was performed (Fig. 7B) and the radioactivity in the different fractions measured (Fig. 7C). Radioactivity was associated to a fraction corresponding to the mobility of PMA3, indicating that the MtmMII methyltransferase converts 9-DPMA3 into PMA3.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Methylation activity of the MtmMI methyltransferase. A, time course for the methylation using as substrate 4-DPMC. open circles, S. lividans containing pWHM3 plus 4-DPMC; closed circles, S. lividans MJM1 minus 4-DPMC; open triangles, S. lividans MJM1 plus 4-DPMC. B, HPLC analysis of the products of the reaction. C, measurement of radioactivity in the different HPLC fractions.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Methylation activity of the MtmMII methyltransferase. A, time course for the methylation using as substrate 9-DPMA3. Open circles, S. lividans containing pWHM3 plus 9-DPMA3; closed circles, S. lividans MJM2 minus 9-DPMA3; open triangles, S. lividans MJM2 plus 9-DPMA3. B, HPLC analysis of the products of the reaction. C, measurement of radioactivity in the different HPLC fractions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During biosynthesis of the antitumor drug mithramycin three methylation events occur. Two of them will modify the architecture of the polyketide moiety and involve a 4-O-methylation and a 9-C-methylation of the premithramycinone and premithramycin precursors, respectively. The third one is a C-methylation during the biosynthesis of D-mycarose, the third deoxysugar present in the trisaccharide chain. Experiments shown in this paper demonstrate that the products of the mtmMI and mtmMII genes are responsible for catalyzing the first two methylation reactions. The MtmMI methyltransferase would convert 4-DPMC into PMC. This assertion is based on two lines of experimental evidence: (i) the major compound accumulated by the M3MI mutant was 4-DPMC and (ii) 4-DPMC was efficiently used as substrate by the MtmMI methyltransferase and was converted into PMC. This reaction is an important event in the mithramycin biosynthesis since the introduction of a methyl group into the 4-position of the aglycon is essential in order to proceed further on through the glycosylation steps and other methylation and oxygenation events. This conclusion can be deduced from the analysis of compounds accumulated by M3MI mutant, which only accumulates 4-DPMC. In this mutant, genes involved in deoxysugar biosynthesis and sugar transfer to the aglycon are not affected. Therefore, it can be inferred that the introduction of the first D-olivose moiety of the trisaccharide at C-12a-O (PMC to PMA1), catalyzed by glycosyltransferase MtmGIV, requires the presence of the 4-O-methyl group.

The MtmMII methyltransferase would be responsible for the C-methylation at C-9 position of the precursor aglycon. Analysis of the compounds accumulated by M3MII mutant showed that all of them lacked this methyl group. However, in contrast to the methylation step catalyzed by MtmMI, C-9 methylation is not essential to complete mithramycin biosynthesis. The major compound isolated through insertional inactivation of the mtmMII gene (M3MII mutant; compound a) corresponded to the final molecule mithramycin lacking the methyl group at position 7, 7-DMTM. This implies that in the absence of this methylation all glycosylation steps leading to the transfer of the disaccharide and trisaccharide chains can take place. Moreover, it also implies that the MtmOIV oxygenase responsible for opening the fourth ring of premithramycin B (20) can also act on a 9-demethylated substrate.

Previous studies on the compounds accumulated by mutants in the mtmGI and mtmGII genes (18) suggested that C-9 methylation could probably occur after the attachment of the first D-olivose unit to the aglycon. Accordingly, the substrate for the MtmMII methyltransferase would be PMA1. However, the in vitro enzymatic assays shown in this paper strongly suggest that MtmMII must act at a later stage of the mithramycin biosynthesis, namely by methylating 9-DPMA3.

Taking together previous work on mithramycin biosynthesis (18, 20) and the results shown here, a pathway is proposed for the methylation steps during mithramycin biosynthesis (Fig. 8). 4-DPMC is the first stable intermediate of the mithramycin biosynthesis. This is converted into PMC by methylation at C-4-O through the MtmMI methyltransferase. Then two consecutive glycosylation steps occur generating PMA1 (18) and 9-DPMA2. The latter is a hypothetical compound that has not yet been isolated and should be accumulated by a mutant affected in the glycosyltransferase gene responsible for the incorporation of D-mycarose as the last sugar in the trisaccharide chain. This gene has not been identified so far. After incorporation of D-mycarose, 9-DPMA3 is produced, the compound isolated in this work that serves as the substrate for the MtmMII methyltransferase, generating PMA3. Finally transfer of the disaccharide (18), fourth ring breakage (20), and a ketoreduction step takes place rendering the final compound mithramycin. In addition to this main pathway some side reactions can take place regarding methylation and glycosylation steps. On the one hand, C-9 methylation can occur before the completion of the entire trisaccharide chain. This is suggested from the isolation of 9-C-methylpremithramycin A1³ and PMA2 (18), compounds obtained from the M3G1 and M3G2 mutants (affected in the mtmGI and mtmGII genes, respectively). Conversion of PMA1 into PMA2 (Fig. 8) would require the participation of the MtmMII methyltransferase and a glycosyltransferase (probably the MtmGIII glycosyltransferase; Ref. 23). On the other hand, the C-9 methylation can be bypassed leading to the formation of 7-demethylmithramycin, a novel compound isolated in this work. All the studies on the mithramycin biosynthetic pathway so far suggest that enzymes acting on late biosynthetic steps, i.e. after the formation of the last nonglycosylated intermediate PMC (methyltransfer, glycosyltransfer, and oxygenation steps), show a certain substrate flexibility. This opens up the possibility of using these enzymes for generating molecular biodiversity that could produce novel potentially useful bioactive compounds.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Proposed pathway for the methylation steps in mithramycin biosynthesis (for details on the assignation of functions to the glycosyltransferase genes (see Refs. 18 and 23). All the compounds shown have been isolated except 9-demethylpremithramycin A2.

	FOOTNOTES

^* This work was supported in part by Plan Nacional en Biotecnologia Grant BIO97-0771 (to J. A. S.), European Union Grant BIO4-CT96-0068 (to J. A. S. and J. R.), and the Medical University of South Carolina Institutional Research Funds of 1999-2000 (to J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a predoctoral fellowship of the Spanish Ministery of Education and Science (F.P.I.).

To whom correspondence for chemical communications should be addressed. E-mail: rohrj@musc.edu.

^** To whom correspondence for molecular biology communications should be addressed. Tel. and Fax: 34-985-103652; E-mail: jasf@sauron.quimica.uniovi.es.

² I. Aguirrezabalaga, C. Olano, N. Allende, L. Rodriguez, A. F. Braña, C. Méndez, and J. A. Salas, submitted for publication.

³ E. Fernández, unpublished results.

	ABBREVIATIONS

The abbreviations used are: 4-DPMC, 4-demethylpremithramycinone; 9-DMPA3, 9-demethylpremithramycin A3; 7-DMTM, 7-demethylmithramycin; PMC, premithramycinone; kb, kilobase(s); PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; ORF, open reading frame; PMA1, premithramycin A1; PMA2, premithramycin A2; PMA3, premithramycin A3.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Hopwood, D. A., and Sherman, D. H. (1990) Annu. Rev. Genet. 24, 37-66[CrossRef][Medline] [Order article via Infotrieve]
2.	Summers, R. G., Wendt-Pienkowski, E., Motamedi, H., and Hutchinson, C. R. (1992) J. Bacteriol. 174, 1810-1820[Abstract/Free Full Text]
3.	Decker, H., Motamedi, H., and Hutchinson, C. R. (1993) J. Bacteriol. 175, 3876-3866[Abstract/Free Full Text]
4.	Connors, N. C., Bartel, P. L., and Strohl, W. R. (1990) J. Gen. Microbiol. 136, 1887-1894
5.	Connors, N. C., and Strohl, W. R. (1993) J. Gen. Microbiol. 139, 1353-1362
6.	Madduri, K., Torti, F., Colombo, A. L., and Hutchinson, C. R. (1993) J. Bacteriol. 175, 3900-3904[Abstract/Free Full Text]
7.	Paulus, T. J., Tuan, J. S., Luebke, V. E., Maine, G. T., DeWitt, J. P., and Katz, L. (1990) J. Bacteriol. 172, 2541-2546[Abstract/Free Full Text]
8.	Kreuzman, A. J., Turner, J. R., and Yeh, W. (1988) J. Biol. Chem. 263, 15626-15633[Abstract/Free Full Text]
9.	Tercero, J. A., Espinosa, J. C., Lacalle, R. A., and Jiménez, A. (1996) J. Biol. Chem. 271, 1579-1590[Abstract/Free Full Text]
10.	Fawaz, F., and Jones, G. H. (1988) J. Biol. Chem. 263, 4602-4606[Abstract/Free Full Text]
11.	Kuzuyama, T., Hidaka, T., Kamigiri, K., Imai, S., and Seto, H. (1992) J. Antibiot. 45, 1812-1814[Medline] [Order article via Infotrieve]
12.	Remers, W. A. (1979) The Chemistry of Antitumor Antibiotics , Vol. 1 , pp. 133-175, Wiley-Interscience, New York
13.	Skarbek, J. D., and Speedie, M. K. (1981) in Antitumor Compounds of Natural Origin (Aszalos, A., ed), Vol. 1 , pp. 191-235, CRC Press, Boca Raton, FL
14.	Dewick, P. M. (1997) Medicinal Natural Products: A Biosynthetic Approach , p. 85, John Wiley & Sons, Chichester, New York
15.	Blanco, G., Fu, H., Méndez, C., Khosla, C., and Salas, J. A. (1996) Chem. Biol. 3, 193-196[CrossRef][Medline] [Order article via Infotrieve]
16.	Lombó, F., Blanco, G., Fernández, E., Méndez, C., and Salas, J. A. (1996) Gene (Amst.) 172, 87-91[CrossRef][Medline] [Order article via Infotrieve]
17.	Rohr, J., Weißbach, U., Beninga, C., Künzel, E., Siems, K., Bindseil, K., Prado, L., Lombó, F., Braña, A. F., Méndez, C., and Salas, J. A. (1998) Chem. Commun. 1998, 437-438
18.	Fernández, E., Weißbach, U., Sánchez Reillo, C., Braña, A. F., Méndez, C., Rohr, J., and Salas, J. A. (1998) J. Bacteriol. 180, 4929-4937[Abstract/Free Full Text]
19.	Rohr, J., Méndez, C., and Salas, J. A. (1999) Bioorg. Chem. 27, 41-54[CrossRef]
20.	Prado, L., Fernández, E., Weißbach, U., Blanco, G., Quirós, L. M., Braña, A. F., Méndez, C., Rohr, J., and Salas, J. A. (1999) Chem. Biol. 6, 19-30[CrossRef][Medline] [Order article via Infotrieve]
21.	Fernández, E., Lombó, F., Méndez, C., and Salas, J. A. (1996) Mol. Gen. Genet. 251, 692-698[Medline] [Order article via Infotrieve]
22.	Bullock, W. O., Fernández, J. M., and Short, J. M. (1987) BioTechniques 5, 376
23.	Blanco, G., Fernández, E., Fernández, Ma, J., Braña, A. F., Weibach, U., Rohr, J., Méndez, C., and Salas, J. A. (1999) Mol. Gen. Genet., in press
24.	Vara, J., Lewandowska-Skarbek, M., Wang, Y. G., Donadio, S., and Hutchinson, C. R. (1989) J. Bacteriol. 171, 5872-5881[Abstract/Free Full Text]
25.	Lombó, F., Braña, A. F., Méndez, C., and Salas, J. A. (1999) J. Bacteriol. 181, 642-647[Abstract/Free Full Text]
26.	Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
27.	Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Schrempf, H. (1985) Genetic Manipulation of Streptomyces: A Laboratory Manual , The John Innes Foundation, Norwich, England
28.	Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract/Free Full Text]
29.	Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395
30.	Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
31.	Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
32.	Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., and Hasegawa, M. (1995) Biosci. Biotechnol. Biochem. 59, 1099-1106[Medline] [Order article via Infotrieve]
33.	Dickens, M. L., Ye, J., and Strohl, W. R. (1995) J. Bacteriol. 177, 536-543[Abstract/Free Full Text]
34.	Lacalle, R. A., Ruiz, D., and Jimenez, A. (1991) Gene (Amst.) 109, 55-61[CrossRef][Medline] [Order article via Infotrieve]
35.	Niemi, J., and Mäntsälä, P. (1995) J. Bacteriol. 177, 2942-2945[Abstract/Free Full Text]
36.	Kagan, R. M., and Clarke, S. (1994) Arch. Biochem. Biophys. 310, 417-427[CrossRef][Medline] [Order article via Infotrieve]
37.	Lombó, F., Siems, K., Braña, A. F., Méndez, C., Bindseil, K., and Salas, J. A. (1997) J. Bacteriol. 179, 3354-3357[Abstract/Free Full Text]