3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics
1998; Elsevier BV; Volume: 273; Issue: 11 Linguagem: Inglês
10.1074/jbc.273.11.6030
ISSN1083-351X
AutoresChun-Gyu Kim, Tin‐Wein Yu, Craig B. Fryhle, Sandeep Handa, Heinz G. Floss,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoThe biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an α,β-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in the A. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA. The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an α,β-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in the A. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA. The clinically important ansamycin antibiotic, rifamycin B (Scheme FSI), contains a biosynthetically unique structural element called a mC7N unit (shown in bold in the rifamycin B structure) (1Ghisalba O. Chimia. 1985; 39: 79-88Google Scholar, 2Floss H.G. Beale J.M. Angew. Chem. 1989; 101 (Angew. Chem. Int. Ed. Engl. 28, 146–177): 147-178Crossref Google Scholar). This mC7N unit is derived from 3-amino-5-hydroxybenzoic acid (AHBA) 1The abbreviations used are: AHBA, 3-amino-5-hydroxybenzoic acid; aminoDHS, 5-deoxy-5-amino-3-dehydroshikimic acid; PLP, pyridoxal phosphate; aminoDHQ, 5-deoxy-5-amino-3-dehydroquinic acid; aminoSA, 5-deoxy-5-aminoshikimic acid; aminoDAHP, 3,4-dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate; DHS, 3-dehydroshikimic acid; DAHP, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate; ESI-MS, electrospray ionization mass spectrometry; ORF, open reading frame; PMP, pyridoxamine phosphate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high performance liquid chromatography; bp, base pair(s); kb, kilobase pair(s); GC-MS, gas chromatography-mass spectroscopy. 1The abbreviations used are: AHBA, 3-amino-5-hydroxybenzoic acid; aminoDHS, 5-deoxy-5-amino-3-dehydroshikimic acid; PLP, pyridoxal phosphate; aminoDHQ, 5-deoxy-5-amino-3-dehydroquinic acid; aminoSA, 5-deoxy-5-aminoshikimic acid; aminoDAHP, 3,4-dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate; DHS, 3-dehydroshikimic acid; DAHP, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate; ESI-MS, electrospray ionization mass spectrometry; ORF, open reading frame; PMP, pyridoxamine phosphate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high performance liquid chromatography; bp, base pair(s); kb, kilobase pair(s); GC-MS, gas chromatography-mass spectroscopy. (3Kibby J.J. McDonald I.A. Rickards R.W. J. Chem. Soc. Chem. Commun. 1980; : 768-769Crossref Scopus (37) Google Scholar, 4Anderson M.G. Kibby J.J. Rickards R.W. Rothschild J.M. J. Chem. Soc. Chem. Commun. 1980; : 1277-1278Crossref Scopus (59) Google Scholar, 5Hatano K. Akiyama S. Asai M. Rickards R.W. J. Antibiot. 1982; 35: 1415-1417Crossref PubMed Scopus (55) Google Scholar, 6Anderson M.G. Monypenny D. Rickards R.W. Rothchild J.M. J. Chem. Soc. Chem. Commun. 1989; : 311-313Crossref Google Scholar, 7Ghisalba O. Nüesch J. J. Antibiot. 1981; 34: 64-71Crossref PubMed Scopus (88) Google Scholar), which serves as the starter unit for the assembly of a linear polyketide by addition of acetate and propionate units. The C terminus of the assembled polyketide eventually forms an amide linkage to the amino group of the AHBA moiety to close the macrolactam ring. AHBA, in turn, is generated by a newly discovered biosynthetic reaction sequence, the aminoshikimate pathway, which parallels the first three steps of the shikimate pathway, but is modified by the introduction of nitrogen in the first step (Scheme FSI) (8Kim C.-G. Kirschning A. Bergon P. Ahn Y. Wang J.J. Shibuya M. Floss H.G. J. Am. Chem. Soc. 1992; 114: 4941-4943Crossref Scopus (46) Google Scholar, 9Kim C.-G. Kirschning A. Bergon P. Zhou P. Su E. Sauerbrei B. Ning S. Ahn Y. Breuer M. Leistner E. Floss H.G. J. Am Chem. Soc. 1996; 118: 7486-7491Crossref Scopus (88) Google Scholar) to give 3,4-dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP) instead of the normal shikimate pathway intermediate, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate (DAHP). Cyclization and dehydration leads to the 5-amino analog of 3-dehydroshikimic acid, 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS), which is then aromatized by the enzyme, AHBA synthase. In this article, we report on the purification and preliminary mechanistic analysis of this enzyme, which has no parallel in the normal shikimate pathway; on the cloning, sequence analysis, and expression of the gene encoding it; and on the effect of deletion of this gene on rifamycin production. Fermentation media were purchased from Difco, and radiochemicals ([α-32P]dCTP, [γ-32P]dATP, and [α-35S]dCTP) were from NEN Life Science Products. AminoDHQ, aminoDHS, and [7-13C]AHBA (89 atom% 13C) were samples prepared previously (9Kim C.-G. Kirschning A. Bergon P. Zhou P. Su E. Sauerbrei B. Ning S. Ahn Y. Breuer M. Leistner E. Floss H.G. J. Am Chem. Soc. 1996; 118: 7486-7491Crossref Scopus (88) Google Scholar). Resins for protein purification and MonoQ and Phenyl-Superose prepacked FPLC columns were purchased from Pharmacia Biotech Inc., molecular size standards for SDS-PAGE were from Bio-Rad, and PVDF membrane from Millipore Corp. All commercially available enzymes were purchased from Boehringer Mannheim, U. S. Biochemical Corp., Life Technologies, Inc., or New England Biolabs; the kits for PCR and sequencing were from U. S. Biochemical Corp.; and Escherichia coli XL1-Blue and E. coli XL1-Blue MRF were from Stratagene. BL21(DE3) was obtained from Novagen, and plasmids were purchased from Novagen (pLysS), Stratagene (pSK−), and Invitrogen (pRSET). The following buffers were used in the protein purifications Buffer A consisted of 50 mm Tris·HCl (pH 7.5), 1 mm phenylmethylsulfonyl fluoride, 10% glycerol, 1 mm EDTA, 1 mm dithiothreitol. Buffer B consisted of 100 mm Tris·HCl (pH 7.5), 1 mm phenylmethylsulfonyl fluoride, 10% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 50 mmKCl. Buffer C consisted of 50 mm Tris·HCl (pH 7.5), 1 mm phenylmethylsulfonyl fluoride, 20% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 50 mmKCl. Buffer D consisted of 50 mm Tris·HCl (pH 7.5). UV-visible measurements were made with a Hewlett-Packard 8452A diode array spectrophotometer. GC-MS spectra were recorded on a Hewlett-Packard 5790A/5970 GC-MS instrument. FPLC was carried out on a Pharmacia system equipped with an LCC-500 controller, UV-M monitor, and dual P-500 pumps. DNA sequencing was done with a Hoefer SE1500 Poker Face apparatus. Electrospray ionization mass spectra (ESI-MS) were acquired using a Fisons Trio 2000 mass spectrometer. Amycolatopsis mediterranei S699, a gift from Dr. G. C. Lancini (Lepetit S.A., Geranzano, Italy), was grown as described previously (9Kim C.-G. Kirschning A. Bergon P. Zhou P. Su E. Sauerbrei B. Ning S. Ahn Y. Breuer M. Leistner E. Floss H.G. J. Am Chem. Soc. 1996; 118: 7486-7491Crossref Scopus (88) Google Scholar). To produce cells for enzyme isolation, 10 ml of a production culture was used to inoculate 500 ml of vegetative medium in a 2-liter flask without a spring coil. After incubation for 54 h at 28 °C and a shaker (New Brunswick Scientific Co., G25) speed of 300 rpm, the mycelia were harvested and used to prepare cell-free extracts. AminoDHS solution (500 μl, 0.6 mm) and buffer B were mixed with a portion of the protein solution (50–300 μl) to give a total volume of 1 ml. After 1 h of incubation at 28 °C, the reaction was stopped by the addition of 200 μl of 15% trichloroacetic acid solution. After centrifugation, the production of AHBA was assessed by measuring the increase in A 296 relative to a blank. Protein concentration was determined with Bio-Rad protein assay solution, using bovine serum albumin as a standard. To determine the kinetic parameters of the native or recombinant enzyme, initial velocities were measured by incubating 50 or 100 μl of enzyme solution at 33 °C with 0.1–1.1 mm aminoDHS in buffer C in a total volume of 1 ml and following the absorbance at 310 nm. The reaction was linear for at least 10 min. Wet cells of A. mediterranei (25 g) were suspended in 100 ml of buffer B and disrupted by two passages through a French pressure cell. The cellular homogenate was centrifuged at 39,000 × g for 20 min at 4 °C. The supernatant was adjusted to 50% ammonium sulfate saturation by the slow addition, with stirring on ice, of powdered (NH4)2SO4, followed by stirring for an additional 30 min. The precipitate was discarded. The (NH4)2SO4 concentration was then raised to 70%, and the protein pellet was dissolved in a minimal amount of buffer C and dialyzed for 4, 10, and 10 h against three changes of 40 volumes of the same buffer. Preswollen DE 52 resin was equilibrated in buffer C and packed at a bed height of 8 cm (bed volume of 40 ml) following the manufacturer's protocol. The dissolved 50–70% ammonium sulfate precipitate (15–20 ml, 300–350 mg of protein) was applied to the column at a flow rate of 48 ml/h. After the sample had been loaded, the column was washed until the A 280 was constant. A linear gradient of 50–350 mm KCl in buffer C (total volume of 240 ml) was applied, and fractions of 4 ml were collected. The fractions containing the AHBA synthase activity (fractions 31–38) were pooled (28–32 ml) and dialyzed overnight against buffer C. A Phenyl-Sepharose CL-4B suspension in buffer C containing 30% ammonium sulfate was packed in a 2.5 cm × 10-cm column to a bed height of 8 cm (bed volume 40 ml). The sample (28–32 ml, 90–100 mg of protein) from DE 52 chromatography in buffer C 30% saturated with ammonium sulfate was applied to the column at a flow rate of 48 ml/h. The column was then washed with 40 ml of buffer C 30% saturated with ammonium sulfate, followed by elution of AHBA synthase with a linear gradient of 30–0% ammonium sulfate in buffer C (total volume 160 ml). Fractions of 2 ml were collected, and the fractions containing AHBA synthase (fractions 47–55) were pooled (14–16 ml) and dialyzed for 4 and 10 h against two changes of 40 volumes of buffer C. Sephadex G-200 resin in buffer C containing 150 mm KCl was packed in a column (2.5 cm × 50 cm) at a flow rate of 10 ml/h (bed volume 220 ml). The enzyme solution from the Phenyl-Sepharose column, concentrated to a volume of 2 ml in buffer C containing 150 mm KCl, was applied to the column, which was then eluted with the same buffer at a flow rate of 10 ml/h. Fractions of 3 ml each were collected, and the fractions of high specific activity (fractions 43–48) were combined, concentrated to a volume of 2 ml, and dialyzed for 4 and 10 h against two changes of 40 volumes of buffer C. The AHBA synthase solution (8 mg of protein) from the gel filtration was loaded onto a Mono Q column (bed volume 1 ml). The column was washed with buffer C for 5 min at 0.5 ml/min and then eluted with a linear gradient of 50–250 mmKCl in buffer C for 5 min at 1 ml/min. Fractions were collected every 2 min. Step gradients were continued for 10 min with 250 mmKCl and for 15 min with 1 m KCl at a flow rate of 0.5 ml/min. The fractions containing high AHBA synthase activity, eluting just before 250 mm KCl, were pooled. Ammonium sulfate was added to the AHBA synthase pool from the Mono Q FPLC to give 30% saturation. The solution (1.6 mg of protein) was applied to a Phenyl-Superose FPLC column (bed volume of 1 ml) at 0.5 ml/min. The column was flushed with buffer C containing 30% saturated ammonium sulfate for 3 min at 0.5 ml/min, followed by elution with a linear gradient of 30–0% saturated ammonium sulfate in buffer C over 30 min. Fractions were collected every 2 min, and the fractions containing high AHBA synthase activity, eluting at 20–22 min, were pooled. Protein from this step was subjected to SDS-PAGE, and the band corresponding to AHBA synthase was cut out and transferred electrophoretically to Immobilon PVDF transfer membrane (Millipore Corp.). The membrane was used for gas phase microsequencing at the protein analysis facility of the Department of Biochemistry, University of Washington. DNA sequencing was performed using the Sequenase kit (U. S. Biochemical Corp.) and [α-35S]dCTP (NEN Life Science Products) according to the manufacturer's protocol. Sequencing reactions were analyzed on polyacrylamide gels (8% (v/v) acrylamide, 5% bisacrylamide, 8m urea, 45 mm Tris borate, pH 8.0, and 1 mm EDTA). SK, KS, T3, or T7 primers (Stratagene) for dsDNA sequencing reactions and M13 primer (Stratagene) for ssDNA sequencing reactions were used. ssDNA was prepared using M13K07 (Promega) following the manufacturer's protocol. DNA and protein sequences were analyzed using the University of Wisconsin Genetics Computer Group (UWGCG) program version 7.3 (10Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Google Scholar). A primary disruption of the AHBA synthase gene was engineered using a marker-replacement suicide vector, pSK−/AHBA2. In this plasmid, the hygromycin resistance gene (hyg) of pIJ963 (11Lydiate D.J. Malpartida F. Hopwood D.A. Gene (Amst.). 1985; 35: 223-235Crossref PubMed Scopus (149) Google Scholar), which resides on a 1.75-kbBglII fragment, had been inserted into the 2.3-kbXhoI fragment of pSK−/AHBA1 (Fig. 2 A) at the only BglII site, which lies at the N-terminal part of the AHBA synthase gene, in an orientation such that transcription of the hyg gene would occur in the same direction as that of the AHBA synthase gene, leaving 1.2 and 1.1 kb of homologous DNA flanking this insertion to the left and right, respectively. Through electroporation (12Mazy-Servais C. Baczkowski D. Dusart J. FEMS Microbiol. Lett. 1997; 151: 135-138Crossref PubMed Google Scholar), approximately 20 transformants of A. mediterranei S699 that were resistant to hygromycin were obtained per microgram of heat-treated denatured pSK−/AHBA2. Southern hybridization of the transformants (data not shown) demonstrated that they arose by the expected single crossover, either upstream, named HGF001, or downstream, named HGF002, of the AHBA synthase gene, in approximately equal numbers. HGF001 produced normal rifamycin B yields; however, all HGF002 recombinants showed delayed production and reduced yield of rifamycin B (approximately half the yield of wild-typeA. mediterranei S699). One of the HGF001 strains was chosen for maintenance on synthetic agar medium (13Lancini G. Rehm H.-J. Reed G. Biotechnology. 4. VCH Verlagsgesellschaft, Weinheim, Germany1986: 431-463Google Scholar) lacking hygromycin. After propagation through three subsequent generations, about 0.5–1% of the colonies showed sporulation 1–2 days earlier and lost the ability to produce rifamycin B. Southern hybridization with six random choices of these rifamycin B nonproducing colonies (HGF003–1 to -6) (data not shown) confirmed that pSK−/AHBA2 had integrated into the chromosome in HGF001, and that all six HGF003 strains had undergone the second crossover event to replace the endogenous AHBA synthase gene with one that was truncated with the insertion of the hygromycin resistance gene marker (Fig. 2 B). pSK−/AHBA1, which contains the 2.3-kb XhoI fragment of A. mediterranei DNA carrying the AHBA synthase gene, was digested with EcoRI. The resulting 1.6-kb EcoRI fragment was resolved on 0.8% agarose gel and ligated into pRSET digested with EcoRI using T4 DNA ligase. The ligation products were transformed into BL21(DE3)/pLysS. Several colonies of E. coli BL21(DE3)/pLysS/pRSET(AHBA) grown on LB agar plates containing carbenicillin (50 μg/ml) and chloramphenicol (34 μg/ml) were inoculated into 10 ml of LB medium containing the same antibiotics. After 5–6 h of growth at 37 °C with shaking (280 rpm), 1 ml of these cultures was used to inoculate 100 ml of LB medium containing 1 m sorbitol, 2.5 mm betaine, 50 μg/ml carbenicillin, and 34 μg/ml chloramphenicol. These cultures were incubated at 30 °C until the A 600reached 1.0. Isopropyl-1-thio-β-d-galactopyranoside was then added to a final concentration of 0.1 mm, and the expression level during further incubation was checked by SDS-PAGE and assay of AHBA synthase activity. A 500-ml overnight culture of E. coli BL21(DE3)/pLysS/pRSET(AHBA) in LB medium containing 1 m sorbitol, 2.5 mmbetaine, carbenicillin, and chloramphenicol (A 600 = 1.7) was transferred to 5 liters of the same medium in a 10-liter fermentor. Fermentation was carried out at 30 °C and 230 rpm with an aeration rate of 3 liters/min. At an A 600 of 1.2, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 0.1 mm. Aeration rate and stirrer speed were increased to 6 liters/min and 280 rpm, respectively. The cells were harvested after 24 h of growth (A 600 = 1.7) and washed with buffer D. The washed cells were suspended in 100 ml of buffer A. The cell suspension was passed through a French pressure cell twice and centrifuged. (NH4)2SO4 was added to the supernatant, and the 35–55% precipitate was collected, dissolved in buffer C, and dialyzed against the same buffer overnight. The dialyzed protein solution was loaded onto a column of DE52 anion exchange resin (bed volume 45 ml), pre-equilibrated with buffer C, at a flow rate of 48 ml/h. After washing with one bed volume of buffer C, the column was eluted with a gradient of 50–350 mm KCl in buffer C (total 350 ml). The fractions containing AHBA synthase were collected and resolved on nickel resin (Novagen) following the manufacturer's protocol. The nickel resin was packed into an FPLC column (bed volume of 7 ml) and washed first with three bed volumes of distilled water, second with five volumes of charge buffer (50 mm NiSO4), and finally with three volumes of binding buffer (20 mmTris-HCl, pH 8.0, containing 5 mm imidazole and 500 mm KCl). The protein solution was loaded onto the column at a flow rate of 1 ml/min, which was then washed with 10 volumes of binding buffer and 6 volumes of washing buffer (20 mmTris-HCl, pH 8.0, containing 60 mm imidazole and 500 mm KCl). Bound protein was eluted with a linear gradient of 60–100 mm imidazole over 30 min, with AHBA synthase eluting at about 80 mm imidazole. To a 60-μl sample of purified recombinant AHBA synthase (1 mg of protein/ml, Tris buffer, pH 7.5) were added at room temperature, in 15-min intervals, three portions of a few micrograms each of NaBH4. After 1 h total reaction time, the pH was adjusted to 7.0 by addition of a few microliters of trifluoroacetic acid. The sample as well as a 60-μl sample of unreduced enzyme were desalted by HPLC on a Vydak C-4 column (Rainin) using a linear gradient of water/0.06% trifluoroacetic acid to acetonitrile/0.06% trifluoroacetic acid (1 ml/min over 60 min). The fractions containing the protein (30–35 min) were pooled, lyophilized, and dissolved in 50–60 μl of 60% acetonitrile/40% water/0.06% trifluoroacetic acid. Samples of 20 μl were then introduced into the mass spectrometer at a flow rate of 16 μl/min and data were acquired, averaging over 15–20 scans/sample. Data processing involved the maximum entropy deconvolution (MaxEnt) procedure (14Ferrige A.G. Seddon M.J. Green B.N. Jarvis S.A. Skilling J. Rapid Commun. Mass Spectrom. 1992; 6: 707-711Crossref Scopus (238) Google Scholar, 15Ferrige A.G. Seddon M.J. Skilling J. Ordsmith N. Rapid Commun. Mass Spectrom. 1992; 6: 765-770Crossref Scopus (26) Google Scholar). To 2 mg of purified recombinant AHBA synthase in 0.5 ml of Tris buffer, pH 7.5, was added aminoDHS (1.2 mm) and an excess of tritiated NaBH4 (300 mCi/mmol). After 20 min, the reaction was quenched by addition of 6 n HCl to pH 2. The protein was removed by centrifugation, and the pH was adjusted to 8 with 1n NaOH. The supernatant, which at this point contained about 23 μCi of tritium, was incubated with alkaline phosphatase and then subjected to preparative layer chromatography (silica gel, n-butanol/water/acetic acid 2:1:1). The majority of the radioactivity coincided with authentic aminoSA (RF = 0.43), the reduction product of the free substrate, but a broad band of radioactivity (∼5%) at a higher RF (0.55–0.7) was seen, whereas no radioactivity was evident at the position of authentic 5-deoxy-5-(N-pyridoxylamino)shikimic acid (N-pyridoxyl-aminoSA) (RF = 0.30). The broad high RF band upon rechromatography separated into two bands, one cochromatographing with authenticN-pyridoxyl-AHBA (RF = 0.58) and the other (RF = 0.67) unidentified, but not identical with authentic pyridoxine (RF = 0.44). A reference sample of N-pyridoxyl-AHBA was synthesized by mixing 7.6 mg of AHBA and 13.3 mg of PLP·H2O, each in 0.5 ml of 0.05 m phosphate buffer, pH 7. After 15 min, when according to TLC Schiff's base formation was complete, excess NaBH4 was added and the mixture kept at room temperature for 1 h with occasional shaking. The product was isolated by ion exchange chromatography (Bio-Rad AG 1X8, formate, successive elution with 3 n HCOOH, 0.1 n HCl, and 1 nHCl) and preparative layer chromatography (silica gel, n-butanol/water/acetic acid 2:1:1, RF = 0.26). The resulting N-phosphopyridoxyl-AHBA was then dephosphorylated by incubation with alkaline phosphatase and the N-pyridoxyl-AHBA purified by preparative layer chromatography in the same system. N-Pyridoxyl-aminoSA was prepared analogously from aminoSA and PLP. AHBA synthase was purified from 54-h-old mycelia of A. mediterranei strain S699 as summarized in Table I. The enzyme activity was assayed by measuring the amount of AHBA formed after 1 h incubation of the protein with 0.3 mm aminoDHS at 28 °C, followed by addition of trichloroacetic acid. In the early stages of purification, AHBA was quantitated by an inverse isotope dilution GC-MS assay (9Kim C.-G. Kirschning A. Bergon P. Zhou P. Su E. Sauerbrei B. Ning S. Ahn Y. Breuer M. Leistner E. Floss H.G. J. Am Chem. Soc. 1996; 118: 7486-7491Crossref Scopus (88) Google Scholar), in the later stages by measuring the increase in A 296, the absorption maximum of AHBA at an acidic pH. The six-step 180-fold purification (Table I) gave AHBA synthase of a specific activity of 72 units/mg protein in 5% overall yield. The protein at this stage was judged by SDS-PAGE to be homogeneous (Fig. 1).Table IPurification of AHBA synthase from A. mediterranei S699StepVolumeActivityRecoveryTotal units1-aNanomoles of AHBA produced per minute.Units/mg of proteinml%Crude extract1-bObtained from 25 g wet cells.1104530.450–70% (NH4)2SO416325172 (72)1-cCumulative yield is given in parentheses.DE 52303173.298 (70)Phenyl-Sepharose CL-4B15220669 (49)Sephadex G-200151301759 (29)Mono Q (FPLC)2885568 (19)Phenyl-Superose (FPLC)2227225 (5)1-a Nanomoles of AHBA produced per minute.1-b Obtained from 25 g wet cells.1-c Cumulative yield is given in parentheses. Open table in a new tab The native molecular mass of AHBA synthase was estimated by gel filtration as 74 kDa and by nondenaturing PAGE as slightly higher than that of bovine serum albumin (66,700 Da). Elution of the enzymatically active band from nondenaturing PAGE and reanalysis by SDS-PAGE gave a molecular mass of 39 kDa, suggesting that the native enzyme is a dimer. Kinetic parameters were determined using partially purified enzyme from the DE 52 column step, assaying activity by following the change in A 310 during the initial 10-min linear phase of the reaction. The enzyme is most active at 33 °C and has a pH optimum of 7.5. AHBA synthase retained its activity over a broad range of temperature and pH. Over 84% of the maximum activity of AHBA synthase was maintained over a temperature range from 28 to 50 °C and a pH range from 7.0 to 9.0. The Km value for aminoDHS was determined from Lineweaver-Burk plots as 0.164 mm. Several compounds were tested as substrates of AHBA synthase, using the protein solution from the Mono Q column step. AHBA synthase could not utilize 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), 5-deoxy-5-aminoshikimic acid (aminoSA), quinic acid, 3-dehydroquinic acid, or 3-dehydroshikimic acid (DHS) as substrate. Furthermore, when the substrate, aminoDHS (0.5 mm), was incubated with AHBA synthase and 3,4-dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP, 1 mm), aminoSA (1 mm) or 3-deoxy-d-arabino-heptulosonic acid 7-phosphate (DAHP, 1 mm), none of the compounds except DAHP affected the enzyme activity, with DAHP showing 40 to 50% activation of the enzyme. It had been shown by others (16Gygax D. Christ M. Ghisalba O. Nüesch J. FEMS Microbiol. Lett. 1982; 15: 169-173Google Scholar) that DAHP synthase from A. mediterranei is inhibited by rifamycin. Activation of AHBA synthase by DAHP provides another piece of evidence that the AHBA biosynthetic pathway is related to the shikimate pathway, and suggests some type of cross-regulation of the two pathways. To obtain partial amino acid sequence information for the construction of oligodeoxynucleotide primers for the cloning of the AHBA synthase gene, the protein from the Phenyl-Superose FPLC step was further purified by SDS-PAGE. The specific region containing the AHBA synthase band was cut out from the gel after visualization with Coomassie Blue and electrophoretically transferred to PVDF membrane (17Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar, 18Gershoni J.M. Palade G.E. Anal. Biochem. 1982; 124: 396-405Crossref PubMed Scopus (305) Google Scholar). Gas-phase microsequencing of the intact protein revealed a 10-amino acid N-terminal sequence H2N-N-A-R-K-A-P-E-F-P-A (sequence 1). Internal amino acid sequences were determined after in situ CNBr cleavage (19Simpson R.J. Nice E.C. Biochem. Int. 1984; 8: 787-791PubMed Google Scholar,20Choli T. Kapp U. Wittmann B. J. Chromatog. 1989; 476: 59-72Crossref PubMed Scopus (43) Google Scholar) of the enzyme on the PVDF membrane. The cleavage products were separated by reverse-phase HPLC (21Hermodson M. Mahoney W. Methods Enzymol. 1983; 91: 352-359Crossref PubMed Scopus (56) Google Scholar) on a C4 column, and two peptides were chosen for amino acid sequence analysis. They yielded sequences of 26 and 16 amino acids, R-L-N-E-F-S-A-S-V-L-R-A-Q-L-A-R-L-D-E-Q-I-A-V-R-L-E (sequence 2) and G-V-G-P-G-T-E-V-I-V-P-A-F-T-*-I-S (sequence 3). Based on the above partial amino acid sequences of AHBA synthase, three degenerate oligodeoxynucleotides were designed (TableII), taking into account the preferred codon usage (22Bibb M.J. Janssen G.R. Ward J.M. Gene (Amst.). 1985; 41: E357-E391Crossref Scopus (74) Google Scholar) of genes from organisms with GC-rich DNA, likeA. mediterranei or Streptomyces (>90% G or C in the third base). These oligonucleotides were used as PCR primers to amplify a 500-bp (combination primers 1 and 2) and a 250-bp (combination primers 1 and 3) region of genomic DNA from A. mediterranei S699. Both PCR products hybridized strongly to the same bands of restriction-digested genomic DNA, establishing them to originate from the same region. Sequencing of the two PCR products revealed that the deduced amino acid sequence of the 257-bp PCR product contained the last two amino acids of the N-terminal peptide sequence and the first six amino acids of the internal peptide sequence 3, neither of which had been used in the construction of the primers. On the other hand, the deduced amino acid sequence of the 505-bp PCR product contained the first 13 amino acids of the internal peptide sequence 2, which had not been encoded by the primer, but did not have the N-terminal amino acid sequence corresponding to oligonucleotide 1 at the 5′ end of the PCR product. Instead, the 505-bp PCR product also had the same sequence as the oligonucleotide 2 at the 5′ end, suggesting that oligonucleotide 1 might bind nonspecifically within a region where the AHBA synthase gene is located. Furthermore, none of the deduced amino acid sequence corresponding to the peptide 3 was found in the 505-bp PCR product.Table IIOligonucleotide primers synthesized in this workPrimersDegenerated oligonucleotidesPeptide templatesPrimer 15′-AAC GCY CGY CCY GAG TT-3′Asn-Ala-Arg-Lys-Ala-Pro-Glu-PhePrimer 25′-GAT CTG CTC GTC YAG YCG YGC YAG-3′Leu-Ala-Arg-Leu-Asp-Glu-Gln-IlePrimer 35′-GT GAA YGC YGG YAC GAT YAC CTC-3′Glu-Val-Ile-Val-Pro-Ala-Phe-Thr
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