Cloning, Overexpression, and Purification of Novobiocic Acid Synthetase from Streptomyces spheroides NCIMB 11891
2000; Elsevier BV; Volume: 275; Issue: 28 Linguagem: Inglês
10.1074/jbc.m003066200
ISSN1083-351X
AutoresMarion Steffensky, Shu‐Ming Li, Lutz Heide,
Tópico(s)Enzyme Structure and Function
ResumoNovobiocic acid synthetase, a key enzyme in the biosynthesis of the antibiotic novobiocin, was cloned from the novobiocin producer Streptomyces spheroides NCIMB 11891. The enzyme is encoded by the gene novL, which codes for a protein of 527 amino acids with a calculated mass of 56,885 Da. The protein was overexpressed as a His6 fusion protein inEscherichia coli and purified to apparent homogeneity by affinity chromatography and gel chromatography. The purified enzyme catalyzed the formation of an amide bond between 3-dimethylallyl-4-hydroxybenzoic acid (ring A of novobiocin) and 3-amino-4,7-dihydroxy-8-methyl coumarin (ring B of novobiocin) in an ATP-dependent reaction. NovL shows homology to the superfamily of adenylate-forming enzymes, and indeed the formation of an acyl adenylate from ring A and ATP was demonstrated by an ATP-PPi exchange assay. The purified enzyme exhibited both activation and transferase activity, i.e. it catalyzed both the activation of ring A as acyl adenylate and the subsequent transfer of the acyl group to the amino group of ring B. It is active as a monomer as determined by gel filtration chromatography. The reaction was specific for ATP as nucleotide triphosphate and dependent on the presence of Mg2+ or Mn2+. ApparentK m values for ring A and ring B were determined as 19 and 131 μm, respectively. Of several analogues of ring A, only 3-geranyl-4-hydroxybenzoate and to a lesser extent 3-methyl-4-aminobenzoate were accepted as substrates. Novobiocic acid synthetase, a key enzyme in the biosynthesis of the antibiotic novobiocin, was cloned from the novobiocin producer Streptomyces spheroides NCIMB 11891. The enzyme is encoded by the gene novL, which codes for a protein of 527 amino acids with a calculated mass of 56,885 Da. The protein was overexpressed as a His6 fusion protein inEscherichia coli and purified to apparent homogeneity by affinity chromatography and gel chromatography. The purified enzyme catalyzed the formation of an amide bond between 3-dimethylallyl-4-hydroxybenzoic acid (ring A of novobiocin) and 3-amino-4,7-dihydroxy-8-methyl coumarin (ring B of novobiocin) in an ATP-dependent reaction. NovL shows homology to the superfamily of adenylate-forming enzymes, and indeed the formation of an acyl adenylate from ring A and ATP was demonstrated by an ATP-PPi exchange assay. The purified enzyme exhibited both activation and transferase activity, i.e. it catalyzed both the activation of ring A as acyl adenylate and the subsequent transfer of the acyl group to the amino group of ring B. It is active as a monomer as determined by gel filtration chromatography. The reaction was specific for ATP as nucleotide triphosphate and dependent on the presence of Mg2+ or Mn2+. ApparentK m values for ring A and ring B were determined as 19 and 131 μm, respectively. Of several analogues of ring A, only 3-geranyl-4-hydroxybenzoate and to a lesser extent 3-methyl-4-aminobenzoate were accepted as substrates. 3-dimethylallyl-4-hydroxybenzoic acid 3-amino-4,7-dihydroxy-8-methyl coumarin noviose electron impact mass spectrometry 3-geranyl-4-hydroxybenzoic acid isopropyl-β-d-thiogalactoside kilobase pair(s) National Collection of Industrial, Food and Marine Bacteria nickel-nitrilotriacetic acid polyacrylamide gel electrophoresis polymerase chain reaction high pressure liquid chromatography The aminocoumarin antibiotic novobiocin is produced byStreptomyces spheroides and Streptomyces niveus. Novobiocin (see Fig. 1) consists of three moieties: a prenylated 4-hydroxybenzoic acid (ring A),1 a substituted aminocoumarin moiety (ring B), and a deoxysugar (ring C). Ring A is attached to the amino group of ring B via an amide bond. Both aromatic rings are derived from tyrosine, ring C is derived from glucose, and the prenyl group of ring A is formed via the nonmevalonate pathway (1.Li S.-M. Hennig S. Heide L. Tetrahedron Lett. 1998; 39: 2717-2720Crossref Scopus (32) Google Scholar, 2.Steffensky M. Li S.-M. Vogler B. Heide L. FEMS Microbiol. Lett. 1998; 161: 69-74Crossref Google Scholar, 3.Kominek L.A. Sebek O.K. Dev. Ind. Microbiol. 1974; 15: 60-69Google Scholar). The antimicrobial activity of novobiocin results from its interaction with bacterial DNA gyrase, which has been investigated by x-ray crystallographic studies (4.Maxwell A. Trends Microbiol. 1997; 5: 102-109Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 5.Lewis R.J. Singh O.M.P. Smith C.V. Skarzynski T. Maxwell A. Wonacott A.J. Wigley D.B. EMBO J. 1996; 15: 1412-1420Crossref PubMed Scopus (305) Google Scholar, 6.Celia H. Hoermann L. Schultz P. Lebeau L. Mallouh V. Wigley D.B. Wang J.C. Mioskowski C. Oudet P. J. Mol. Biol. 1994; 236: 618-628Crossref PubMed Scopus (34) Google Scholar, 7.Wigley D.B. Davies G.J. Dodson E.J. Maxwell A. Dodson G. Nature. 1991; 351: 624-629Crossref PubMed Scopus (486) Google Scholar). The detailed knowledge available about the structural elements of novobiocin involved in its binding to the biological target may permit rational approaches in the search for new aminocoumarin derivatives. Recently, the development of new, synthetic aminocoumarin compounds with gyrase-inhibiting activity has been reported (8.Ferroud D. Collard J. Klich M. Dupuis-Hamelin C. Mauvais P. Lassaigne P. Bonnefoy A. Musicki B. Bioorg. Med. Chem. Lett. 1999; 9: 2881-2886Crossref PubMed Scopus (45) Google Scholar, 9.Laurin P. Ferroud D. Schio L. Klich M. Dupuis-Hamelin C. Mauvais P. Lassaigne P. Bonnefoy A. Musicki B. Bioorg. Med. Chem. Lett. 1999; 9: 2875-2880Crossref PubMed Scopus (54) Google Scholar, 10.Laurin P. Ferroud D. Klich M. Dupuis-Hamelin C. Mauvais P. Lassaigne P. Bonnefoy A. Musicki B. Bioorg. Med. Chem. Lett. 1999; 9: 2079-2084Crossref PubMed Scopus (160) Google Scholar). Like novobiocin itself (Albamycin®, Pharmacia & Upjohn), such new aminocoumarins may serve as antibiotics for the treatment of infections with multi-resistant Gram-positive bacteria such as Staphylococcus aureus or Staphylococcus epidermidis (11.Raad I.I. Hachem R.Y. Abi-Said D. Rolston K.V.I. Whimbey E. Buzaid A.C. Legha S. Cancer. 1998; 82: 403-411Crossref PubMed Scopus (49) Google Scholar, 12.Montecalvo M.A. Horowitz H. Wormser G.P. Seiter K. Carbonaro C.A. Antimicrob. Agents Chemother. 1995; 39: 794Crossref PubMed Scopus (60) Google Scholar, 13.Walsh T.J. Standiford H.C. Reboli A.C. John J.F. Mulligan M.E. Ribner B.S. Montgomerie J.Z. Goetz M.B. Mayhall C.G. Rimland D. Stevens D.A. Hansen S.L. Gerard G.C. Ragual R.J. Antimicrob. Agents Chemother. 1993; 37: 1334-1342Crossref PubMed Scopus (121) Google Scholar). Genetic engineering and combinatorial biosynthesis in bacteria provide an important new tool for drug discovery. Besides polyketide synthetases, peptide synthetases especially have been successfully used for such approaches (14.Bently R. Bennet J.W. Annu. Rev. Microbiol. 1999; 53: 411-446Crossref PubMed Scopus (76) Google Scholar, 15.Khosla C. Gordon E.M. Kerwin J.F. Combinatorial Chemistry and Molecular Diversity in Drug Discovery. Wiley-Liss, Inc., New York1998: 401-417Google Scholar, 16.Mootz H.D. Marahiel M.A. Curr. Opinion Biotech. 1999; 10: 341-348Crossref PubMed Scopus (55) Google Scholar, 17.Symmank H. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 21581-21588Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Knowledge of the sequence and function of the genes involved in the biosynthesis of natural products is a prerequisite for such research. We have recently cloned and sequenced the biosynthetic gene cluster for novobiocin from S. spheroides NCIMB 11891 and have assigned functions to the biosynthetic genes by comparison with GenBankTM entries and by gene inactivation experiments (18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google Scholar). A key step in the biosynthesis of novobiocin is the formation of the amide bond between ring A and ring B (Fig. 1) in an ATP-dependent reaction; this enzymatic reaction, termed novobiocic acid synthetase, has been demonstrated previously in crude extracts from a novobiocin-producing strain (19.Kominek L.A. Meyer H.F. Methods Enzymol. 1975; 43: 502-508Crossref PubMed Scopus (12) Google Scholar). A detailed investigation of this reaction is of particular interest for the development of new aminocoumarin antibiotics; whereas ring B and ring C are essential for the binding of novobiocin to gyrase, the structure of ring A can be varied without loss of antibiotic activity (10.Laurin P. Ferroud D. Klich M. Dupuis-Hamelin C. Mauvais P. Lassaigne P. Bonnefoy A. Musicki B. Bioorg. Med. Chem. Lett. 1999; 9: 2079-2084Crossref PubMed Scopus (160) Google Scholar, 20.Althaus I.W. Dolak L. Reusser F. J. Antibiotics. 1988; 41: 373-376Crossref PubMed Scopus (21) Google Scholar). It has been suggested that the structure of ring A influences the uptake of the antibiotic through the bacterial membrane (20.Althaus I.W. Dolak L. Reusser F. J. Antibiotics. 1988; 41: 373-376Crossref PubMed Scopus (21) Google Scholar, 21.Reusser F. Dolak L.A. J. Antibiotics. 1986; 39: 272-274PubMed Google Scholar). Cloning of the gene(s) for novobiocic acid synthetase and investigation of the substrate specificity of this reaction may therefore assist in the development of novobiocin derivatives with a modified ring A. We now report the cloning, overexpression, purification, and characterization of novobiocic acid synthetase from S. spheroides NCIMB 11891, encoded by the gene novL. [32P]Tetrasodium pyrophosphate (126.4 GBq/mmol) was obtained from NEN Life Science Products. Ring B and novobiocic acid were kindly provided by Pharmacia & Upjohn, Inc. (Kalamazoo, MI). 3-Cyclohexyl-4-hydroxybenzoic acid was a gift from L. Wessjohann (Amsterdam, Netherlands). Plicatin B was kindly provided by R. Bates (Bangkok, Thailand). Ring A was obtained by hydrolysis of novobiocin as described previously (19.Kominek L.A. Meyer H.F. Methods Enzymol. 1975; 43: 502-508Crossref PubMed Scopus (12) Google Scholar). 3-Dimethylallyl-4-hydroxycinnamic acid was synthesized by hydrolysis of plicatin B as described in Bates et al. (22.Bates R.W. Gabel C.J. Ji J. Rama-Devi T. Tetrahedron. 1995; 51: 8199-8212Crossref Scopus (43) Google Scholar); EI-MS analysis on a TSQ70 spectrometer (Finnigan, Bremen, Germany) using methanol as solvent confirmed the identity of the product (observed molecular weight, 232.2; theoretical molecular weight of C14H16O3, 232.3). 3-Geranyl-4-hydroxybenzoic acid (GBA) was synthesized enzymatically from 4-hydroxybenzoic acid and geranyl diphosphate with 4-hydroxybenzoate polyprenyltransferase of Escherichia coli(23.Melzer M. Heide L. Biochim. Biophys. Acta. 1994; 1212: 93-102Crossref PubMed Scopus (99) Google Scholar); the incubation mixture (3 ml) contained 0.4 mm4-hydroxybenzoic acid, 2 mm geranyl diphosphate, 50 mm MgCl2, 1 mm KF, 50 mm Tris-HCl buffer, pH 8.0, and 0.2 mg/ml membrane protein fraction and was incubated for 60 min at 37 °C. The reaction was stopped by addition of 90 μl of concentrated formic acid. GBA was extracted with 30 ml of n-hexan, the organic phase was evaporated, and the residue was dissolved in 50 mm Tris-HCl buffer, pH 8.0. The bacterial strains and plasmids used in this study are listed in Table I. Plasmid pUWL201 was kindly provided by A. Bechthold (Tübingen, Germany) and originally obtained from U. Wehmeier (Wuppertal, Germany). Cloning experiments were performed in E. coli XL1 Blue MRF′ by standard procedures (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Heterologous expression experiments withStreptomyces lividans TK24 were carried out as described previously (18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google Scholar). Enzyme activity was determined after cultivation in CDM medium (20 μg/ml thiostrepton) for 3–4 days at 28 °C and 170 rpm in baffled shake flasks. DNA manipulations and standard genetic techniques in E. coli and Streptomyces species were carried out as described in Sambrook et al. (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and Hopwood et al. (25.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. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. The John Innes Foundation, Norwich, UK1985Google Scholar).Table IBacterial strains and plasmids used in this studyStrain or plasmidDescriptionReference or sourceS. spheroides NCIB 11891Wild type, novoblocin producerNCIMBS. lividansTK24Streptomycin-resistant, no plasmidsRef. 25.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. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. The John Innes Foundation, Norwich, UK1985Google ScholarE. coli XL1 Blue MRF′TetrStratageneE. coli BL21 (DE3)pLysSCamrNovagenpBluescript SK(−) (pBSK(−))AmprStratagenepGEM-11Zf(+)AmprPromegapQE70T5 promotor, C-terminal His6 tag, AmprQlagenpRSet BT7 promotor, N-terminal His6 tag, AmprInvitrogenpUWL201Ampr Tsrr ermE up promotorU. Wehmeier aUnpublished data.p9–6GE99.7-kb EcoRI fragment from comid 9–6G ofS. spheroides cosmid library in pBSK(−)Ref. 18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google ScholarpMS651.95-kb EcoRI-BglII fragment (novH) of p9–6GE9 in pUWL201Ref. 18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google ScholarpMS736.96-kb EcoRI-XbaI fragment (novH, I, J, K, andL) of p9–6GE9 in pUWL201Ref. 18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google ScholarpMS762.1-kbApaI fragment (novL) of p9–6GE9 in pGEM-11Zf(+)This workpMS772.1-kbEcoRI-XbaI fragment (novL) of pMS76 in pUWL201This workpMS781.95-kbEcoRI-BglII fragment of p9–6GE9 in pMS76 (novH and novI)This workpMS794.08-kbEcoRI-XbaI fragment (novH andnovI) of pMS78 in pUWL201This workpMS80novL in pQE70This workpMS82novL in pRSet BThis worka Unpublished data. Open table in a new tab The streptomyces expression vector pUWL201, containing theermE up promoter, was used for the construction of the expression plasmids. For the expression of novL, a 2.1-kbApaI fragment from plasmid p9–6GE9 containingnovL was cloned into the same site of pGEM-11Zf(+) to give pMS76. Restriction analysis confirmed that the EcoRI site of pGEM-11Zf(+) was located upstream of novL. The insert of pMS76 was excised with EcoRI and XbaI and ligated into the same sites of pUWL201 to give pMS77. pMS65, containing novH as a 1.95-kbEcoRI-BglII fragment in expression vector pUWL201, has been described previously (18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google Scholar). pMS79, containing bothnovH and novL in pUWL201, was prepared by ligation of the same 1.95-kb EcoRI-BglII fragment carrying novH into theEcoRI-BamHI-sites of pMS76. Both genes in the resulting plasmid pMS78 were oriented in the same direction. The 4.08-kb insert of pMS78 was excised with EcoRI andXbaI and cloned into the same sites of pUWL201 to yield pMS79. Expression constructs were transformed into S. lividans TK24 and examined for novobiocic acid synthetase activity as described previously (18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google Scholar). novL was amplified by PCR using pMS76 DNA as template. An SphI site was introduced at the place of the natural start codon, using primer novL-1 (5′-TAGCCACGCATGCCGAACAAGGATCAC-3′; bold letters represent the SphI site). At the C terminus, a BamHI site was introduced before and an EcoRI site behind the stop codon, using primer novL-3 (5′-CATCGAATTCTCAGGATCCCCTGTCCACCA-3′; bold letters represent introduced restriction sites). The PCR mixture (100 μl) contained 100 ng of pMS76 template, 22 pmol of each primer, 0.2 mm dNTPs (Stratagene), Pfu DNA polymerase reaction buffer, and 5% (v/v) Me2SO. 2.5 units of clonedPfu DNA polymerase (Stratagene) were added after an initial denaturation for 5 min at 96 °C, followed by 27 cycles (95 °C for 90 s, 72 °C for 45 s, and 72 °C for 4 min). The PCR product was digested with SphI and BamHI before ligation into the same sites of the expression vector pQE70, resulting in a C-terminal in-frame fusion with the His6 tag of pQE70. The resulting plasmid was designated as pMS80. novL was again amplified by PCR. A BglII site was introduced at the N-terminal side for in-frame ligation to the His6 tag of pRSet B using primer novL-4 (5′-CGAAAGATCTCCACATATGGCGAACAAGG-3′). The GTG start codon of novL was changed to an ATG codon by introduction of a NdeI site, which offers the possibility to remove the sequence for the N-terminal His tag by NdeI digestion and religation. Primer novL-3 (see above) was again used as C-terminal primer. PCR was carried out as described above, with an annealing temperature of 65 °C. TheBglII-EcoRI-digested PCR product was ligated into the same sites of pRSet B to create pMS82. The use of primer novL-3 resulted in the extension of the C-terminal end of the encoded protein from the original -VDR to -VDRGS. Expression experiments with pMS82 were performed in E. coli BL21(DE3)pLysS cultivated in NZYCM broth (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 50 μg/ml carbenicillin and 34 μg/ml chloramphenicol at 37 °C. E. coli XL1 Blue MRF′ was used as host for the expression of pMS80. Cells were cultured at 30 °C in LB medium (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 50 μg/ml carbenicillin until anA 600 of 0.7 was reached. 0.5 mmisopropyl-β-d-thiogalactoside (IPTG) was added, and after further growth for 3 h at 30 °C, cells were harvested by centrifugation and washed with 50 mm Tris-HCl, pH 8.0. All subsequent steps were carried out at 4 °C. Cells (3 g) were suspended in 3 ml of lysis buffer (50 mmNaH2PO4, pH 8.0, 300 mm NaCl, 10 mm imidazole, 1 mg/ml lysozyme). After incubation on ice for 30 min, the cell suspension was sonicated for 1 min (Branson Sonifier 250). 10 μg/ml RNase A and 5 μg/ml DNase I were added, and the mixture was incubated on ice for further 10 min. After removal of cellular debris by centrifugation (17,500 × g for 30 min), 1 ml of Ni-NTA-agarose slurry (50% (w/v) nickel-nitrilotriacetic acid agarose resin suspension in 30% (v/v) ethanol, precharged with Ni2+) (Qiagen) were added and mixed gently by shaking for 60 min. The lysate-Ni-NTA-agarose mixture was loaded into a column. Unbound proteins were removed by washing with 8 ml of wash buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 20 mm imidazole), and the NovL fusion protein was eluted with 2 ml of elution buffer (50 mmNaH2PO4, pH 8.0, 300 mm NaCl, 250 mm imidazole). The Ni-NTA-agarose eluate was applied to a HiLoad 26/60 Superdex 200 column (Amersham Pharmacia Biotech) that had been equilibrated with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm dithiothreitol, and 50 μm phenylmethylsulfonyl fluoride. Chromatography was carried out with the same buffer at a flow rate of 1.5 ml/min. Fractions were assayed for novobiocic acid synthetase activity. Protein concentrations were determined by the Bradford method (26.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar) using bovine serum albumin as a standard. SDS-PAGE was carried out according to the method of Laemmli (27.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar), and protein bands were stained with Coomassie Brilliant Blue R-250. The molecular weight of native NovL was determined by gel filtration on a HiLoad 26/60 Superdex 200 column using the buffer described above. The column was calibrated with blue dextran 2000, aldolase (molecular weight, 158,000), albumin (molecular weight, 67,000), ovalbumin (molecular weight, 43,000), and ribonuclease A (molecular weight, 13,700) (Amersham Pharmacia Biotech). The novobiocic acid synthetase assay contained 1 mm ring A, 1 mmring B, 5 mm ATP, 5 mm MnCl2, and 50 mm Tris-HCl, pH 8.0, in a final volume of 100 μl. To assay the activity of crude extracts, 20–100 μg of protein and an incubation time of 20 min were used. To assay the activity of purified novobiocic acid synthetase, a maximum of 5 μg of enzyme and an incubation time of 7 min were used to ensure linearity of the product formation. The reaction was carried out at 30 °C and stopped by addition of 5 μl of 1.5 m trichloroacetic acid. The reaction mixture was extracted with 1 ml of ethyl acetate, the organic phase was evaporated, and the residue was dissolved in H2O/methanol (50:50, v/v). HPLC analysis was carried out using a Multosphere RP18–5 column (250 × 4 mm, 5 μm; C + S Chromatographie Service, Düren, Germany) with a linear gradient from 60 to 100% methanol in 1% aqueous formic acid and detection at 305 nm. Authentic novobiocic acid was used as a standard. The reaction mixture of the ATP-PPi exchange assay (28.Stachelhaus T. Mootz H.D. Bergendahl V. Marahiel M.A. J. Biol. Chem. 1998; 273: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) contained (in a final volume of 100 μl) 50 mm Tris-HCl, pH 8.0, 5 mm MnCl2, 5 mm ATP, 0.1 mm tetrasodium pyrophosphate, 34.4 kBq [32P]tetrasodium pyrophosphate, 4 μg of purified novobiocic acid synthetase, and 1 mm of the tested substrates ring A or ring B. After incubation for 20 min at 30 °C, the reaction was stopped by adding 1 ml of a mixture containing 1.2% (w/v) activated charcoal, 0.1 m tetrasodium pyrophosphate, and 3% (v/v) perchloric acid. The charcoal was pelleted by centrifugation (14,000 rpm for 5 min), washed twice with 1 ml of water, and finally resuspended in 0.5 ml of water. The charcoal-bound radioactivity was measured using a Tri-Carb 2100TR scintillation analyzer (Canberra-Packard) after addition of 9 ml of liquid scintillation fluid (Rotiszint® Eco Plus, Roth). To confirm the identity of the product of the novobiocic acid synthetase reaction, 2 ml (22.5 mg of protein) of a crude extract fromS. lividans TK24 transformed with the expression construct pMS77 were passed through a Sephadex G-25 column and incubated in 50 mm Tris-HCl, pH 8.0, with 1 mm ring A, 1 mm ring B, 5 mm ATP, and 5 mmMnCl2 in a final volume of 15 ml for 60 min at 30 °C. After addition of 750 μl of 1.5 m trichloroacetic acid, the incubation mixture was extracted for three times with 20 ml of ethyl acetate. The organic phases were combined, evaporated, and dissolved in H2O/methanol (50:50, v/v), and the reaction product was purified by HPLC as described for the novobiocic acid synthetase assay (see above). EI-MS was carried out as described under "Chemicals and Radiochemicals" using dichloromethane as a solvent. A molecular weight of 395.2 was observed (for novobiocic acid, C22H21 NO6, the molecular weight was 395.4). To confirm the identity of the product formed from the ring A analogue GBA, 50 μg of purified NovL, 0.5 mm GBA, 1 mmRing B, 5 mm ATP, and 5 mm MnCl2were incubated in a final volume of 1 ml in 50 mm Tris-HCl, pH 8.0 for 20 min at 30 °C. The reaction was stopped by addition of 50 μl of 1.5 m trichloroacetic acid. After extraction with 3 × 1 ml of ethyl acetate and evaporation of the organic phase, the residue was dissolved in in H2O/methanol (50:50 v/v), and the reaction product was purified by HPLC as described for the novobiocic acid synthetase assay (see above). EI-MS analysis was performed as described under "Chemicals and Radiochemicals," and a molecular weight of 463.3 was observed (theoretical result: molecular weight of C27H29 NO6 was 463.5). Sequence analysis of the novobiocin biosynthetic gene cluster revealed two genes for which homology searches suggested a possible involvement in the novobiocic acid synthetase reaction (18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google Scholar); the deduced protein sequence of novH showed similarity to nonribosomal peptide synthetases, and novL showed homology to acyl-CoA synthetases and 4-coumarate:CoA ligases. Novobiocic acid synthetase activity could be demonstrated (18.Steffensky M Mühlenweg A. Wang Z.-X. Li S.-M. Heide L. Antimicrob. Agents Chemother. 2000; 44: 1214-1222Crossref PubMed Scopus (180) Google Scholar) upon heterologous expression of a 6.96 kb DNA fragment comprising novH,novL, and three further complete open reading frames (Fig.2, pMS73). To identify the genes involved in this reaction, we now prepared additional constructs containing these genes, expressed them in S. lividans TK24, and examined the resulting novobiocic acid synthetase activity. All constructs were derived from pUWL201 and contained the ermE up promoter for foreign gene expression. Whereas expression of novH (Fig. 2,pMS65) yielded no activity, novobiocic acid synthetase was clearly detected upon expression of novL (pMS77). The identity of the enzymatic product was confirmed by HPLC in comparison with authentic substance and by preparative isolation of the product followed by mass spectroscopy (EI-MS; see "Experimental Procedures"). Simultaneous expression of novH andnovL (pMS79) did not increase activity in comparison to the expression of novL alone (Fig. 2), and likewise a mixture of the two enzyme extracts obtained after separate expression of genesnovH and novL (pMS65 and pMS77), respectively, did not show higher activity than the extract obtained from the expression of novL alone (data not shown). This demonstrates that the novobiocic acid synthetase reaction is catalyzed by NovL alone and that NovH is not required for this activity. Within the novobiocin biosynthetic gene cluster (GenBankTMaccession number AF170880), novL spans positions 12457–14040. It comprises 1584 base pairs and encodes a protein of 527 amino acids (calculated mass, 56,885 Da). The coding region has an overall G + C content of 70.1%. Upstream of the GTG initiation codon, a putative ribosomal binding site (AGGTAG) was identified. Fig.3 shows that NovL contains several conserved motifs supposed to be involved in common steps of adenylate formation, i.e. nucleotide binding, PPi release, and adenylation of the carboxylate moiety of the substrate. These motifs include Box I (SSGTTGXPKGV) and a sequence similar to the Box II motif (usually GEICIRG) of 4-coumarate:CoA ligases (29.Stuible H.-P. Büttner D. Ehlting J. Hahlbrock K. Kombrink E. FEBS Lett. 2000; 467: 117-122Crossref PubMed Scopus (93) Google Scholar), as well as motifs A8 and A10 in the C-terminal domain (30.Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (901) Google Scholar). As confirmed by mutational analysis, both the conserved Lys of the Box I motif and the conserved Arg of motif A8 cooperate in coordinating the pyrophosphate release during adenylate formation (29.Stuible H.-P. Büttner D. Ehlting J. Hahlbrock K. Kombrink E. FEBS Lett. 2000; 467: 117-122Crossref PubMed Scopus (93) Google Scholar). The conserved Lys of motif A10 interacts with the carboxyl group of the substrate as well as with the ribose oxygens O-4′ and O-5′ (30.Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (901) Google Scholar). Becker-André et al. (31.Becker-Anrdré M. Schulze-Lefert P. Hahlbrock K. J. Biol. Chem. 1991; 266: 8551-8559Abstract Full Text PDF PubMed Google Scholar) suggested a participation of the central cysteine within Box II motif of 4-coumarate:CoA ligases in thiolester formation, but this hypothesis was recently disproven by mutational analysis (29.Stuible H.-P. Büttner D. Ehlting J. Hahlbrock K. Kombrink E. FEBS Lett. 2000; 467: 117-122Crossref PubMed Scopus (93) Google Scholar). A distinction between coenzyme A ligases and enzymes that merely form acyl adenylates is therefore not possible from sequence data. In contrast to nonribosomal peptide synthetases, NovL did not show a 4′-phosphopantetheinyl attachment site. For further characterization, NovL was expressed in E. coli in form of different fusion proteins with a His6 residue for metal affinity chromatography (see "Experimental Procedures"). Fusion of the His tag to the N terminus of NovL resulted in the expression of an unsoluble protein, and only minimal novobiocic acid synthetase activity (0.74 pkat/mg protein) was detectable in the soluble fraction. Fusion of the His tag to the C terminus of NovL gave approximately 7-fold higher activities (5.1 pkat/mg). SDS-PAGE analysis showed, after induction with IPTG, the formation of a 60.6-kDa protein (calculated mass, 58.1 kDa) (Fig. 4), most of which was still in the insoluble fraction. The amount of soluble protein could be increased, however, by decreasing the growth temperature to 30 °C, reducing IPTG concentration to 0.5 mm, induction at a later stage of the growth phase (A 600 = 0.7), and shortening of the induction period to 3 h. Under these conditions, soluble novobiocic acid synthetase was obtained in an activity of 461 pkat/mg protein. The fusion protein of NovL with the C-terminal His6 tag was purified by metal affinity c
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