FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1979353605> ?p ?o ?g. }

• W1979353605 endingPage "30703" @default.
• W1979353605 startingPage "30695" @default.
• W1979353605 abstract "S-Adenosylmethionine:nocardicin 3-amino-3-carboxypropyltransferase catalyzes the biosynthetically rare transfer of the 3-amino-3-carboxypropyl moiety fromS-adenosylmethionine to a phenolic site in the β-lactam substrates nocardicin E, F, and G, a late step of the biosynthesis of the monocyclic β-lactam antibiotic nocardicin A. Whereas a number of conventional methods were ineffective in purifying the transferase, it was successfully obtained by two complementary affinity chromatography steps that took advantage of the two substrate-two product reaction scheme. S-Adenosylhomocysteine-agarose selected enzymes that utilize S-adenosylmethionine, and a second column, nocardicin A-agarose, specifically bound the desired transferase to yield the enzyme as a single band of 38 kDa on a silver-stained SDS-polyacrylamide gel. The transferase is active as a monomer and exhibits sequential kinetics. Further kinetic characterization of this protein is described and its role in the biosynthesis of nocardicin A discussed. The gene encoding this transferase was cloned from a sublibrary of Nocardia uniformis DNA. Translation gave a protein of deduced mass 32,386 Da which showed weak homology to small molecule methyltransferases. However, three correctly disposed signature motifs characteristic of these enzymes were observed. S-Adenosylmethionine:nocardicin 3-amino-3-carboxypropyltransferase catalyzes the biosynthetically rare transfer of the 3-amino-3-carboxypropyl moiety fromS-adenosylmethionine to a phenolic site in the β-lactam substrates nocardicin E, F, and G, a late step of the biosynthesis of the monocyclic β-lactam antibiotic nocardicin A. Whereas a number of conventional methods were ineffective in purifying the transferase, it was successfully obtained by two complementary affinity chromatography steps that took advantage of the two substrate-two product reaction scheme. S-Adenosylhomocysteine-agarose selected enzymes that utilize S-adenosylmethionine, and a second column, nocardicin A-agarose, specifically bound the desired transferase to yield the enzyme as a single band of 38 kDa on a silver-stained SDS-polyacrylamide gel. The transferase is active as a monomer and exhibits sequential kinetics. Further kinetic characterization of this protein is described and its role in the biosynthesis of nocardicin A discussed. The gene encoding this transferase was cloned from a sublibrary of Nocardia uniformis DNA. Translation gave a protein of deduced mass 32,386 Da which showed weak homology to small molecule methyltransferases. However, three correctly disposed signature motifs characteristic of these enzymes were observed. S-adenosyl-l-methionine S-adenosyl-l-homocysteine distilled, deionized water 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate high pressure liquid chromatography polyacrylamide gel electrophoresis kilobase pair S-adenosylmethionine:nocardicin 3-amino-3-carboxypropyltransferase transferase oligonucleotide probe polymerase chain reaction nucleotides. Nocardicin A (see 1 in Fig. 1) is the most biologically active of a series of monocyclic β-lactam antibiotics isolated from the fermentation broth of the actinomycete Nocardia uniformis subsp. tsuyamanesis (ATCC 21806) (1Aoki H. Sakai H. Kohsaka M. Konomi T. Hosoda J. Kubochi Y. Iguchi E. Imanaka H. J. Antibiot. 1976; 29: 492-500Crossref PubMed Scopus (233) Google Scholar, 2Hashimoto M. Komori T. Kamiya T. J. Am. Chem. Soc. 1976; 98: 3023-3025Crossref PubMed Scopus (127) Google Scholar, 3Hashimoto M. Komori T. Kamiya T. J. Antibiot. 1976; 29: 890-901Crossref PubMed Scopus (73) Google Scholar). Co-occurring with the major metabolite 1 are the structurally related monocyclic β-lactams nocardicin B–G (Fig. 1, 2-7) (2Hashimoto M. Komori T. Kamiya T. J. Am. Chem. Soc. 1976; 98: 3023-3025Crossref PubMed Scopus (127) Google Scholar, 4Hosoda J. Konomi T. Tani N. Aoki H. Imanaka H. Agric. Biol. Chem. 1977; 41: 2013-2020Crossref Scopus (3) Google Scholar, 5Kurita M. Jomon K. Komori T. Miyairi N. Aoki H. Kuge S. Kamiya T. Imanaka H. J. Antibiot. 1976; 29: 1243-1245Crossref PubMed Scopus (22) Google Scholar, 6Watanabe K. Okuda T. Yokose K. Furumai T. Maruyama H.B. J. Antibiot. 1983; 36: 321-324Crossref PubMed Scopus (21) Google Scholar). Whole-cell incorporation experiments have shown 1 to be derived from the l-isomers of methionine, serine, and p-hydroxyphenylglycine (PHPG) (8), a degradation product of l-tyrosine. These are the most direct primary precursors of the homoseryl, β-lactam, and aryl portions of the antibiotic, respectively (7Townsend, C. A., and Salituro, G. M. (1984) J. Chem. Soc. Chem. Commun. 1631–1632Google Scholar, 8Townsend C.A. Brown A.M. J. Am. Chem. Soc. 1983; 105: 913-918Crossref Scopus (80) Google Scholar, 9Townsend C.A. Brown A.M. J. Am. Chem. Soc. 1982; 104: 1748-1750Crossref Scopus (23) Google Scholar, 10Townsend C.A. Brown A.M. J. Am. Chem. Soc. 1981; 103: 2873-2874Crossref Scopus (26) Google Scholar, 11Hosoda J. Tani N. Konomi T. Ohsawa S. Aoki H. Imanaka H. Agric. Biol. Chem. 1977; 41: 2007-2012Crossref Scopus (2) Google Scholar). In an important result, it was demonstrated that the simplest member of the nocardicin family, nocardicin G (7), is a direct biosynthetic precursor to nocardicin A (12Townsend C.A. Wilson B.A. J. Am. Chem. Soc. 1988; 110: 3320-3321Crossref Scopus (18) Google Scholar). With nocardicin G (7) positioned as the central intermediate in the pathway leading to nocardicin A (1), the skeleton of the antibiotic can be completed, in unspecified order, by amine oxidation and 3-amino-3-carboxypropyl side chain attachment (Fig. 2). The stereochemical course of side chain attachment was shown to be inversion through the incorporation of methionine stereospecifically deuterated at C-4 (13Townsend, C. A., Reeve, A. M., and Salituro, G. M. (1988) J. Chem. Soc. Chem. Commun. 1579–1581Google Scholar). This configurational outcome parallels the stereochemistry of addition of the 3-aminopropyl moiety derived from decarboxylatedS-adenosylmethionine (AdoMet)1 operative in the biosynthesis of the polyamines (14Golding B.T. Nassereddin I.K. J. Am. Chem. Soc. 1982; 104: 5817-5915Crossref Scopus (17) Google Scholar, 15Golding, B. T., and Nassereddin, I. K. (1985) J. Chem. Soc. Perkin Trans. I 2017–2024Google Scholar, 16Orr G.R. Danz D.W. Pontoni G. Prabhakaran P.C. Gould S.J. Coward J.K. J. Am. Chem. Soc. 1988; 110: 5799-5971Crossref Scopus (31) Google Scholar), as well as more conventional methyltransferases (17Arigoni D. CIBA Found. Symp. 1978; 60: 243-261Google Scholar, 18Floss H.G. Lee S. Acc. Chem. Res. 1993; 26: 116-122Crossref Scopus (55) Google Scholar), suggesting a role for AdoMet in nocardicin A biosynthesis in vivo. This postulate was borne out in preliminary cell-free studies in which an efficient, time-dependent conversion from nocardicin E (5) to nocardicin A in the presence of AdoMet was demonstrated (19Wilson B.A. Bantia S. Salituro G.M. Reeve A.M. Townsend C.A. J. Am. Chem. Soc. 1988; 110: 8238-8239Crossref Scopus (12) Google Scholar). Assuming the epimerization of the amino acid terminus (C-9′) of nocardicin A is a late step in the biosynthesis, the product of this transferase enzyme was expected to be isonocardicin A (L at C-9′). Subsequently, it was demonstrated that an epimerase capable of equilibrating the stereochemistry at C-9′ of nocardicin A was also present in the cell-free extract (19Wilson B.A. Bantia S. Salituro G.M. Reeve A.M. Townsend C.A. J. Am. Chem. Soc. 1988; 110: 8238-8239Crossref Scopus (12) Google Scholar). The bioconversion of nocardicin E (5) to isonocardicin A in cell-free extracts lends support for pathway A (Fig. 2) to nocardicin A (1) in which the amine of nocardicin G (7) is oxidized to the oxime to form nocardicin E (5) and, in the penultimate step, the 3-amino-3-carboxypropyl side chain is attached to the phenolic hydroxy group. The final step, in analogy to penicillin biosynthesis (20Roach P.L. Clifton I.J. Hensgens C.M.H. Shibata N. Schofield C.J. Hajdu J. Baldwin J.E. Nature. 1997; 387: 827-830Crossref PubMed Scopus (392) Google Scholar) and demonstrated experimentally (see below), is then the epimerization of the amino acid terminus to thed-configuration. Although rare, the transfer of the 3-amino-3-carboxypropyl group from AdoMet to a nucleophilic acceptor is not unprecedented. It has been demonstrated in the biosynthesis of the X-base from Escherichia coli tRNAPhe (21Nishimura S. Taya Y. Kuchino Y. Ohashi Z. Biochem. Biophys. Res. Commun. 1974; 57: 702-708Crossref PubMed Scopus (64) Google Scholar), the Y-base from yeast tRNAPhe (22Münch H.-J. Thiebe R. FEBS Lett. 1975; 51: 257-258Crossref PubMed Scopus (21) Google Scholar), the germination inhibitor discadenine from the cellular slime mold Dictyostelium discoideum (23Taya Y. Tanaka Y. Nishimura S. FEBS Lett. 1978; 89: 326-328Crossref PubMed Scopus (25) Google Scholar), and plant siderophores of the mugineic acid family (24Shojima S. Nishizawa N.-K. Mori S. Plant Cell Physiol. 1989; 30: 673-677Google Scholar, 25Shojima S. Nishizawa N.-K. Fushiya S. Nozoe S. Irifune T. Mori S. Plant Physiol. (Bethesda). 1990; 93: 1497-1503Crossref PubMed Scopus (204) Google Scholar). AdoMet has also been shown (26Chen J.-Y.C. Bodley J.W. J. Biol. Chem. 1988; 263: 11692-11696Abstract Full Text PDF PubMed Google Scholar) to be the donor of the 3-amino-3-carboxypropyl unit in diphthamide, the post-translationally modified histidine of elongation factor 2. The only one of the enzymes responsible for this group transfer that has been purified (27Higuchi K. Kanazawa K. Nishizawa N.-K. Chino M. Mori S. Abadía J. Iron Nutrition in Soils and Plants. Kluwer Academic Publishers, The Netherlands1995: 29-35Crossref Google Scholar) is nicotianamine synthase, but this enzyme catalyzes a transformation distinct from the enzyme in the present work and has not been characterized kinetically or mechanistically. A 420-fold partial purification has been reported for discadenine synthase, but the final preparation contained many contaminating proteins (28Ihara M. Tanaka Y. Yanagisawa K. Taya Y. Nishimura S. Biochim. Biophys. Acta. 1986; 881: 135-140Crossref Scopus (5) Google Scholar). In this work, the first purification to apparent homogeneity, kinetic characterization, and cloning of a 3-amino-3-carboxypropyltransferase from a bacterial source is reported. In the presence of AdoMet this enzyme catalyzes the transformation of the substrates nocardicin E, F, and G to the products isonocardicin A, B, and C, respectively. By using reverse genetic techniques, the gene encoding the transferase was cloned from N. uniformis DNA. Translation revealed a 32,389-Da protein containing three modestly conserved motifs characteristic of S-adenosylmethionine-binding sites (29Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310 (; Correction (1995) Arch. Biochem. Biophys. 316, 657): 417-427Crossref PubMed Scopus (423) Google Scholar). Except where noted, all chromatographic matrices, AdoMet (chloride salt) and its derivatives, and reagents for protein purification and affinity column synthesis were products of Sigma. Superose 6 HR 10/30 was a product of Amersham Pharmacia Biotech. Trifluoroacetic acid was purchased from Aldrich. Pre-packed Bio-Gel P6DG desalting columns (10 ml) and dye reagent concentrate for the Bradford (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217317) Google Scholar) protein concentration assay were purchased from Bio-Rad. Centricon-30® (Amicon) microconcentrators were used as directed at 3000 × g and 4 °C. Solution dialysis used Spectra-Por 12–14,000 molecular weight cut-off dialysis membrane (Spectrum Medical Industries). Wild type N. uniformis subsp.tsuyamanesis (ATCC 21806) was purchased as a lyophilized pellet from the ATCC (Rockville, MD). All other chemicals were of reagent grade. Nocardicin substrates and peptides were prepared in this laboratory according to the procedures of Salituro and Townsend (31Salituro G.M. Townsend C.A. J. Am. Chem. Soc. 1990; 112: 760-770Crossref Scopus (58) Google Scholar). Solutions for SDS-PAGE were made according to Laemmli (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207461) Google Scholar). Electrophoresis was run on a Hoeffer Mighty Small II Slab Gel Electrophoresis SE 250 unit using a Hoeffer Scientific Instruments PS500X DC power supply operating at a constant current of 20 mA. Kinetic data for reactions conducted under pseudo-first order conditions were analyzed using non-linear regression computer programs developed in this laboratory (33McGuire S.M. Silva J.C. Casillas E.G. Townsend C.A. Biochemistry. 1996; 35: 11470-11486Crossref PubMed Scopus (30) Google Scholar). Kinetic data for the initial velocity pattern and inhibition studies were fit to the appropriate equation with the FORTRAN programs of Cleland (34Cleland W.W. Methods Enzymol. 1979; 63: 103-108Crossref PubMed Scopus (1929) Google Scholar) to obtain the desired kinetic parameters. Restriction endonucleases were purchased from either Stratagene (La Jolla, CA) or New England Biolabs (Beverly, MA). Cloned PfuDNA polymerase, E. coli XL1 Blue MRF′ cells, T4 polynucleotide kinase, pBluescript II SK (−), ethidium bromide, and nitrocellulose membranes were supplied by Stratagene. T4 DNA ligase was obtained from New England Biolabs. Shrimp alkaline phosphatase and α-35S-dATP and [γ-32P]ATP were bought from Amersham Pharmacia Biotech. Deoxynucleotides were acquired from Perkin-Elmer. Bacto-agar, yeast extract, soluble starch, and Bacto-tryptone were purchased from Difco. Qiagen columns were procured from Qiagen (Chatsworth, CA). The Prep-A-Gene DNA isolation isolation kit was bought from Bio-Rad. The following were provided by Life Technologies, Inc.: agarose, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside, ultra-pure urea, acrylamide, and N, N′-methylenebisacrylamide. Isopropyl-1-thio-β-d-galactopyranoside was purchased from 5′ → 3′, Inc. (Boulder, CO). Tetramethylammonium chloride was obtained from Sigma. Kodak Biomax MR film and X-Omat film were bought from Kodak. Custom-designed oligonucleotides were synthesized on an Applied Biosystems 380B DNA Synthesizer (Foster City, CA), and peptide sequencing analyses were conducted on an Applied Biosystems 470A gas-phase sequencer (Protein/Peptide/DNA Facility, Department of Biological Chemistry, The Johns Hopkins Medical School). HPLC separations were carried out on a Waters 600 HPLC and 490 Programmable Multi-wavelength Detector equiped with a Vydac C4reversed phase column (250 × 10 mm; Heperia, CA). Seed medium (4Hosoda J. Konomi T. Tani N. Aoki H. Imanaka H. Agric. Biol. Chem. 1977; 41: 2013-2020Crossref Scopus (3) Google Scholar), 30 ml, was added to 125-ml Erlenmeyer flasks that were outfitted with a cotton stopper and covered with aluminum foil. Fermentation medium (4Hosoda J. Konomi T. Tani N. Aoki H. Imanaka H. Agric. Biol. Chem. 1977; 41: 2013-2020Crossref Scopus (3) Google Scholar) was prepared with the addition of 2 ml/liter of trace minerals (35Adye J. Mateles R.I. Biochim. Biophys. Acta. 1964; 86: 418-420Crossref PubMed Scopus (233) Google Scholar) and aliquoted in 100-ml portions into 500-ml Erlenmeyer flasks. Media were autoclaved at 20 atm/125 °C for 30 min. Seed flasks were inoculated from a spore suspension, introducing 100 μl into each of five flasks with a sterile pipette tip. The spore suspension was prepared from a natural variant of wild type N. uniformis that had been selected for high nocardicin A production. The seed flasks were maintained at 28 °C and 300 rpm in an incubator shaker. After 48 h, 5 ml of the cell suspension was used to initiate each fermentation flask. Growth continued for 48 h under the same conditions as for the seed flasks. N. uniformis subsp.tsuyamanesis (ATCC 21806), 1 liter of total fermentation volume, was harvested by centrifugation (9,200 × g, 10 min, 4 °C). The cell paste was washed with sterile 0.9% NaCl, centrifuged as above, and suspended in 0.05 m sodium phosphate, pH 7.4, containing 1 mm benzamidine, 0.1 mm phenylmethylsulfonyl fluoride, and 25% glycerol (buffer P) in a 250-ml steel beaker (2 ml of buffer per g wet weight of cells). Cells were ruptured at 0 °C by ultrasonication (Bransonic, model W-225R) at 50% duty cycle, power level 7, for 5 min and centrifuged (16, 300 × g, 30 min, 4 °C). The supernatant was used directly as the cell-free extract. Nucleic acids were precipitated by slowly adding 5% (v/v) polyethyleneimine in buffer P to a final concentration of 0.3% (v/v), stirring 20 min at 0 °C, and centrifuging as for the cell-free extract. Ammonium sulfate was added to the resulting solution in portions over 45 min to a final saturation of 60%. The extract was stirred for an additional 30 min and centrifuged as above. The protein pellets were redissolved in 30 ml of buffer P containing 0.05 mm EDTA (dialysis buffer) and dialyzed overnight versus 2 liters of the same. The dialyzed solution from above was diluted to 300 ml with dialysis buffer and loaded at a flow rate of 1 ml/min onto a 5-ml column of AdoHcy-agarose. The column was washed with 25 ml of the same buffer and eluted with 50 ml of buffer P containing 1 mm AdoMet at the same flow rate. Subsequent chromatographic steps required the inclusion of 1 mm AdoMet in the buffer for activity to be retained. A 5-ml aliquot of the eluate from the AdoHcy column was loaded directly onto a 0.8-ml column of nocardicin A-agarose equilibrated in buffer P containing 1 mm AdoMet. After washing the column with 2 ml of the same buffer, it was eluted with 5 ml of buffer P containing 1 mm AdoMet, 1 mm nocardicin A, and 1 m NaCl. This solution of homogeneous protein was concentrated using two Centricon-30® concentrators to a final volume of about 1 ml. The combined concentrate was desalted on a prepacked column of Bio-Gel P6DG (10 ml) equilibrated in buffer P containing 1 mm AdoMet. A Varian 5020 microprocessor-controlled liquid chromatograph equipped with a 250 × 4.6 mm Spherex (Phenomenex) 5-μm C-18 reversed phase column was used for HPLC assays. Column effluent was monitored by UV spectroscopy at 270 nm on an ABI 1000S Diode Array detector interfaced with a Waters 745 Data Module recorder/integrator. Solvents were HPLC grade and filtered through a 0.45-μm filter prior to use. Water was deionized, distilled, and filtered. To 100 μl of protein solution in a 1.5-ml microcentrifuge tube was added 2 μl of nocardicin E solution (2.9 mg/ml in distilled, dd H2O, final concentration 130 μm) and 8 μl of AdoMet solution (6.6 mg/ml in dd H2O, final concentration 1100 μm). After incubation for 20 min at 37 °C, 10 μl of 1 n HCl was added to stop the reaction. If a precipitate was observed, the assay tubes were centrifuged at 16,000 × g for 5 min. The mixture (100 μl) was injected onto the HPLC column which was eluted isocratically with 0.2% aqueous trifluoroacetic acid, 15% acetonitrile at 1.0 ml/min. AdoMet eluted close to the solvent front, nocardicin A had at R = 5.9 min, and nocardicin E eluted after 8.5 min. Analyses were conducted by HPLC as described with assay time and protein concentration adjusted to produce less than 15% turnover of the substrate in question. If the required dilution produced a protein concentration below 0.1 mg/ml, the enzyme solution was diluted with buffer P containing 1 mg/ml bovine serum albumin in order to avoid dilution inactivation. All substrates were prepared as w/v solutions in H2O with the exception of AdoMet which was assessed spectrophotometrically. Assays were stopped by the addition of 0.1 volume of 1 m HCl, flash-frozen in liquid nitrogen, and stored at −78 °C until analyzed by HPLC. The amount of product produced in the enzymatic reaction (nanomole) was calculated by multiplying the numerical value obtained from the electronic integration by a factor that was determined from a calibration curve. Where nocardicin G was the substrate (and isonocardicin C was the product), the reaction was monitored at 230 nm using 0.05 mammonium acetate as the mobile phase. Commerical AdoMet was purified prior to use by preparative HPLC utilizing a Regis Val-U-Pak 10-μm C-18 column (1 × 25 cm) equilibrated with 0.2% aqueous trifluoroacetic acid. Under these conditions, AdoMet was eluted from the column, whereas the impurities AdoHcy and 5′-methylthioadenosine remained bound. The resulting solution of AdoMet was lyophilized. AdoHcy-agarose-purified transferase solution (5.0 ml) was incubated with nocardicin E (1 mm) and AdoMet (2 mm) at 37 °C for 18 h. The nocardicin product of the reaction was isolated by preparative HPLC (Phenomenex 10-μm ODS, 4 × 25 cm, 8 ml/min, 0.1% aqueous trifluoroacetic acid, 15% CH3CN; v/v) and lyophilized. The purified nocardicin was dissolved in GITC reaction solvent (49.6% H2O, 50% CH3CN, 0.4% Et3N) and derivatized with GITC (2 eq in CH3CN) at 37 °C for 30 min. The derivatized product was analyzed by HPLC (36Kinoshita T. Kasahara Y. Nimura N. J. Chromatogr. 1982; 210: 77-81Crossref Scopus (157) Google Scholar). AdoHcy-agarose was made from 6-aminohexanoic acid-agarose according to the manufacturer's directions. 6-Aminohexanoic acid agarose (5 ml of gel, 11.4 μmol of aminocaproate per ml) was washed with 500 ml of H2O and placed in a 50-ml beaker. AdoHcy (46 mg, about 119 μmol) was suspended in 2 ml of H2O and solubilized with 1 n HCl. After adjusting the pH above 2.0 with saturated NaHCO3, the AdoHcy solution was added to the gel suspension. Water-soluble carbodiimide (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; 100 mg, 0.5 mmol) was added, and the mixture was swirled gently at room temperature to link the carboxylate of the solid support to the amine of AdoHcy. The pH was maintained between 4.5 and 6.0 for 1 h followed by incubation at room temperature overnight. The gel was washed with 500 ml each of water, 1 m NaCl, and water. The column was stored in the presence of 0.4% (w/v) NaN3 in distilled, deionized water at 4 °C until needed. Using ε259 = 15,300, it was calculated that 95 μmol of AdoHcy washed through; therefore, 24 μmol was attached for an efficiency of 20% and a ligand loading of 4.8 μmol/ml gel. N-Hydroxysuccinimide (310 mg, 2.7 mmol, 1.2 eq), bromoacetic acid (312 mg, 2.25 mmol, 1 eq), and dicylohexylcarbodiimide (510 mg, 2.5 mmol, 1.1 eq) were added to 20 ml of p-dioxane in a 50 ml round-bottomed flask. After 1 h at room temperature, the dicyclohexylurea was removed by filtration and the filtrate was evaporated to dryness. The residue was taken up in CH2Cl2, filtered, and evaporated several times to ensure the dicyclohexylurea was removed. After drying under high vacuum for 2 h, the residue was crystallized from CH2Cl2/petroleum ether to yield 425 mg (80%) of active ester as white needles, which was used immediately. NMR (CDCl3) δ = 4.13 (s, 2H, CH2Br); 2.89 (s, 4H, ring). The stoichiometry for gel coupling was 66 μmol of amino groups per 1.5 mmol of active ester. About 300 mg of crystalline active ester was dissolved in 3 ml of p-dioxane. Following filtration, the solution was added to ω-aminohexyl-agarose (8.8 ml, 66.3 μmol of amino groups) pre-equilibrated in 0.1 m sodium phosphate, pH 7.5, to yield a total volume of 15 ml. After standing for 1 h at 4 °C, the gel was washed with 1 liter of 0.1 m NaCl, also at 4 °C. The washed gel was then equilibrated with 200 ml of 0.1m NaHCO3, pH 9.0 (NaOH). Nocardicin A (100 mg, 190 μmol) was dissolved in about 5 ml of 0.1 m NaHCO3, pH 9.0, and added to the gel suspension. After incubating for 3 days at room temperature with gentle shaking, the gel was washed with 2 liters of 0.2 m NaCl at 4 °C. The washed gel was equilibrated with the bicarbonate buffer above (200 ml) and placed in a 50-ml filter flask. After degassing under vacuum, the suspension was reacted with 5 ml of 0.2 m β-mercaptoethanol in the same buffer. The suspension was incubated for 1 h at room temperature and washed with 1 liter of 0.2 m NaCl. Using ε272 = 16,320, it was calculated that 170 μmol of nocardicin A washed through; therefore, 20.5 μmol was attached for an efficiency of 11% (based on excess nocardicin A) and a ligand loading of 2.33 μmol/ml gel. The gel was stored in the presence of 0.04% (w/v) NaN3 in distilled, deionized water at 4 °C until needed. NAT from the nocardicin A-agarose column was purified further by reversed phase HPLC on a Vydac C4 column. After pre-equilibration of the column with 0.1% aqueous trifluoroacetic acid, NAT was injected, and after a 5-min wash with the same solvent, was eluted with an acetonitrile:water gradient (0–60%, each component containing 0.1% trifluoroacetic acid) over 30 min. NAT purified by nocardicin A-agarose column chromatography was treated with Lys-C endoproteinase as described by the supplier (Sigma). The resulting peptide fragments were separated by reversed phase HPLC on a Vydac C4 column. After pre-equilibration with 0.1% aqueous trifluoroacetic acid, peptide fragments were eluted with an acetonitrile:water gradient (0–90%, each component containing 0.1% trifluoroacetic acid) over 90 min. Peptide 1-23-22 was further purified by a second gradient (5–35%) over 60 min, and similarly peptide 1-30-40 was further purified with a gradient (10–40%) over 60 min. The purified peptides were sequenced. High molecular mass (≥50 kb) N. uniformis gDNA was prepared using the cetyltrimethylammonium bromide, large scale bacterial genomic DNA procedure described by Ausubel et al. (39Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar). RNA was removed by an overnight incubation at 37 °C with DNase-free RNase A (final concentration 40 μg/ml). Amplification of the 5′ end of the NAT gene utilized one primer designed from the N-terminal sequence (1-30-40NQ) and two primers designed from the internal Lys-C peptide sequence (1-23-22CE and 1-23-22CD). Degeneracy of all three primers was minimized by taking advantage of the reported Nocardia codon preferences (40Coque J. Malumbres M. Martı́n J.F. Liras P. FEMS Microbiol. Lett. 1993; 110: 91-96Crossref Scopus (9) Google Scholar). A typical 100-μl reaction in 1× cloned Pfu buffer contained 200 ng of N. uniformis gDNA, 250 nmol of each primer, 200 μmol of each dNTP, and 5% glycerol. The reaction mixture was overlaid with mineral oil, denatured for 5 min at 95 °C, and, upon the addition of 2.5 units of cloned Pfu polymerase, was subjected to 30 cycles (94 °C for 3 min and 72 °C for 1 min) and one cycle of (94 °C for 1 min and 72 °C for 10 min) in an Eppendorf Microcycler (Fremont, CA). PCR products were separated on a non-denaturing polyacrylamide gel. The desired band was excised, cloned into SmaI-digested pBluescript II SK(−), and transformed into E. coli XL 1 Blue MRF′ cells. Recombinant pBluescript II SK(−) was purified and sequenced. A non-degenerate oligonucleotide derived from the PCR-amplified 5′ end of the gene encoding NAT (transferase oligonucleotide probe (TOP), TAC GAC CTG TTC TTC CTG) hybridized to a single 6.3-kb PstI fragment in 3 m tetramethylammonium chloride, 50 mmNaPO4, pH 6.8, 0.2% SDS, and 5× Denhardt's solution at 52 °C on a dried agarose gel (41Mather M.W. Keightley J.A. Fee J.A. Methods Enzymol. 1993; 218: 695-704Crossref PubMed Scopus (8) Google Scholar, 42Jacobs K.A. Rudersdorf R. Neill S.D. Dougherty J.P. Brown E.L. Fritsch E.F. Nucleic Acids Res. 1988; 16: 4637-4650Crossref PubMed Scopus (100) Google Scholar, 43Wood W.I. Gitschier J. Lasky L.A. Lawn R.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1585-1588Crossref PubMed Scopus (588) Google Scholar, 44Wallace R.B. Miyada C.G. Methods Enzymol. 1987; 152: 432-442Crossref PubMed Scopus (134) Google Scholar). Accordingly, 30 μg of N. uniformis gDNA was digested with 500 units of PstI at 37 °C for 12 h followed by a 1-h incubation at 37 °C with an additional 100 units of enzyme. Using standard procedures, the digested DNA was precipitated, washed with 70% ethanol, and dissolved in 45 μl of dd H2O (39Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar). The digested DNA was resolved on a 0.6% agarose gel and the DNA between 6 and 9 kb was excised and isolated. The isolated PstI fragments were ligated intoPstI-digested, shrimp alkaline phosphatase-treated pBluescript II SK(−). The ligation product mixture was transformed into E. coli XL 1 BlueMRF′ cells and grown on LB agar containing 90 μg/ml ampicillin, overlaid with isopropyl-1-thio-β-d-galactopyranoside and 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside at 37 °C. Recombinant colonies (200) were regrown on nitrocellulose filters and screened by hybridization with the end-labeled32P-TOP, as described above for the genomic digest. A single colony was isolated that contained a 6.3-kb insert, pSDB 05-152. Sequencing of PCR products was accomplished using Sequenase-2.0 DNA polymerase as described by U. S. Biochemical Corp. using either the appropriate PCR primer or universal primers. The DNA sequence of pSDB 05-152 was obtained by primer walking using automated fluorescent sequencing methods. Sequence data were edited and compiled in Sequencher™ (Gene Codes Corp., Ann Arbor, MI) and analyzed with the GCG software package (Version 8, Genetics Computer Group, Madison, WI). Data base searches were performed online with BLAST 2 at the National Center for Biotechnology Information (45Karlin S. Altschul S.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5873-5877Crossref PubMed Scopus (303) Google Scholar, 46Karlin S. Altschul S.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2264-2268Crossref PubMed Scopus (1189) Google Scholar, 47Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed" @default.
• W1979353605 created "2016-06-24" @default.
• W1979353605 creator A5029061064 @default.
• W1979353605 creator A5063672044 @default.
• W1979353605 creator A5067252545 @default.
• W1979353605 date "1998-11-01" @default.
• W1979353605 modified "2023-09-26" @default.
• W1979353605 title "Purification, Characterization, and Cloning of an S-Adenosylmethionine-dependent 3-Amino-3-carboxypropyltransferase in Nocardicin Biosynthesis" @default.
• W1979353605 cites W110457946 @default.
• W1979353605 cites W1484115349 @default.
• W1979353605 cites W1486157350 @default.
• W1979353605 cites W1528573160 @default.
• W1979353605 cites W1600286284 @default.
• W1979353605 cites W1605786392 @default.
• W1979353605 cites W1606158156 @default.
• W1979353605 cites W1608086343 @default.
• W1979353605 cites W1705731201 @default.
• W1979353605 cites W1817247642 @default.
• W1979353605 cites W1965998064 @default.
• W1979353605 cites W1968120233 @default.
• W1979353605 cites W1975571966 @default.
• W1979353605 cites W1978414804 @default.
• W1979353605 cites W1980265640 @default.
• W1979353605 cites W1981217765 @default.
• W1979353605 cites W1983525937 @default.
• W1979353605 cites W1986063867 @default.
• W1979353605 cites W1986815695 @default.
• W1979353605 cites W1990433107 @default.
• W1979353605 cites W1991972900 @default.
• W1979353605 cites W1994162276 @default.
• W1979353605 cites W1994441123 @default.
• W1979353605 cites W1994762770 @default.
• W1979353605 cites W1997271047 @default.
• W1979353605 cites W1997974036 @default.
• W1979353605 cites W2003206991 @default.
• W1979353605 cites W2008288225 @default.
• W1979353605 cites W2016003384 @default.
• W1979353605 cites W2019808954 @default.
• W1979353605 cites W2022002419 @default.
• W1979353605 cites W2022818851 @default.
• W1979353605 cites W2026701781 @default.
• W1979353605 cites W2031797010 @default.
• W1979353605 cites W2032413102 @default.
• W1979353605 cites W2033932745 @default.
• W1979353605 cites W2038035294 @default.
• W1979353605 cites W2050728905 @default.
• W1979353605 cites W2055275842 @default.
• W1979353605 cites W2059337630 @default.
• W1979353605 cites W2059813020 @default.
• W1979353605 cites W2063543271 @default.
• W1979353605 cites W2063933626 @default.
• W1979353605 cites W2066971343 @default.
• W1979353605 cites W2068607115 @default.
• W1979353605 cites W2068850736 @default.
• W1979353605 cites W2072150399 @default.
• W1979353605 cites W2074868165 @default.
• W1979353605 cites W2074963449 @default.
• W1979353605 cites W2075955253 @default.
• W1979353605 cites W2076024397 @default.
• W1979353605 cites W2078476027 @default.
• W1979353605 cites W2078515702 @default.
• W1979353605 cites W2085863052 @default.
• W1979353605 cites W2091153733 @default.
• W1979353605 cites W2093128077 @default.
• W1979353605 cites W2094519647 @default.
• W1979353605 cites W2095594022 @default.
• W1979353605 cites W2100837269 @default.
• W1979353605 cites W2109122403 @default.
• W1979353605 cites W2116446934 @default.
• W1979353605 cites W2128518196 @default.
• W1979353605 cites W2148464225 @default.
• W1979353605 cites W2153767651 @default.
• W1979353605 cites W2158714788 @default.
• W1979353605 cites W2167520506 @default.
• W1979353605 cites W2201823543 @default.
• W1979353605 cites W2215901593 @default.
• W1979353605 cites W2221878956 @default.
• W1979353605 cites W2225045400 @default.
• W1979353605 cites W2231965155 @default.
• W1979353605 cites W2234008479 @default.
• W1979353605 cites W2338134387 @default.
• W1979353605 cites W2338151994 @default.
• W1979353605 cites W2342242267 @default.
• W1979353605 cites W2342656934 @default.
• W1979353605 cites W2410969779 @default.
• W1979353605 cites W2949200221 @default.
• W1979353605 cites W2951825058 @default.
• W1979353605 cites W2969273703 @default.
• W1979353605 cites W4238270754 @default.
• W1979353605 cites W4293247451 @default.
• W1979353605 cites W1569957410 @default.
• W1979353605 cites W2085848900 @default.
• W1979353605 doi "https://doi.org/10.1074/jbc.273.46.30695" @default.
• W1979353605 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9804844" @default.
• W1979353605 hasPublicationYear "1998" @default.
• W1979353605 type Work @default.
• W1979353605 sameAs 1979353605 @default.
• W1979353605 citedByCount "37" @default.

• next