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• W2008321700 abstract "Isochorismate pyruvate-lyase (IPL), the second enzyme of pyochelin biosynthesis and the product of thepchB gene, was purified to homogeneity fromPseudomonas aeruginosa. In the reaction catalyzed by this enzyme, isochorismate → salicylate + pyruvate, no cofactors appear to be required. At the pH optimum (pH 6.8), the enzyme displayed Michaelis-Menten kinetics, with an apparent Km of 12.5 μm for isochorismate and akcat of 106 min−1, calculated per monomer. The native enzyme behaved as a homodimer, as judged by molecular sieving chromatography, electrophoresis under nondenaturing conditions, and cross-linking experiments. PchB has approximately 20% amino acid sequence identity with AroQ-class chorismate mutases (CMs). Chorismate was shown to be converted to prephenate by purified PchBin vitro, with an apparent Km of 150 μm and a kcat of 7.8 min−1. An oxabicyclic diacid transition state analog and well characterized inhibitor of CMs competitively inhibited both IPL and CM activities of PchB. Moreover, a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, was functionally complemented by the cloned P. aeruginosa pchB gene for growth in minimal medium. A mutant form of PchB, in which isoleucine 88 was changed to threonine, had no detectable IPL activity, but retained wild-type CM activity. In conclusion, the 11.5-kDa subunit of PchB appears to contain a single active site involved in both IPL and CM activity. Isochorismate pyruvate-lyase (IPL), the second enzyme of pyochelin biosynthesis and the product of thepchB gene, was purified to homogeneity fromPseudomonas aeruginosa. In the reaction catalyzed by this enzyme, isochorismate → salicylate + pyruvate, no cofactors appear to be required. At the pH optimum (pH 6.8), the enzyme displayed Michaelis-Menten kinetics, with an apparent Km of 12.5 μm for isochorismate and akcat of 106 min−1, calculated per monomer. The native enzyme behaved as a homodimer, as judged by molecular sieving chromatography, electrophoresis under nondenaturing conditions, and cross-linking experiments. PchB has approximately 20% amino acid sequence identity with AroQ-class chorismate mutases (CMs). Chorismate was shown to be converted to prephenate by purified PchBin vitro, with an apparent Km of 150 μm and a kcat of 7.8 min−1. An oxabicyclic diacid transition state analog and well characterized inhibitor of CMs competitively inhibited both IPL and CM activities of PchB. Moreover, a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, was functionally complemented by the cloned P. aeruginosa pchB gene for growth in minimal medium. A mutant form of PchB, in which isoleucine 88 was changed to threonine, had no detectable IPL activity, but retained wild-type CM activity. In conclusion, the 11.5-kDa subunit of PchB appears to contain a single active site involved in both IPL and CM activity. isochorismate synthase bifunctional enzyme consisting of an N-terminal chorismate mutase and a C-terminal prephenate dehydratase domain as AroQ·PheA but with a C-terminal prephenate dehydrogenase domain chorismate mutase 3,3′-dithiobis(sulfosuccinimidylpropionate) dithiothreitol high performance liquid chromatography isochorismate pyruvate-lyase isopropyl-β-d-thiogalactopyranoside transition state analog Salicylate is a biosynthetic product and a precursor of secondary metabolites and siderophores (iron chelators) in several bacterial genera, e.g. Pseudomonas, Burkholderia, Azospirillum, Vibrio, Yersinia, and Mycobacterium (1Ankenbauer R.G. Cox C.D. J. Bacteriol. 1988; 170: 5364-5367Crossref PubMed Google Scholar, 2Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (150) Google Scholar, 3Sokol P.A. Lewis C.J. Dennis J.J. J. Med. Microbiol. 1992; 36: 184-189Crossref PubMed Scopus (59) Google Scholar, 4Saxena B. Mayuranki M. Modi V.V. J. Gen. Microbiol. 1986; 132: 2219-2224Google Scholar, 5Okujo N. Saito M. Yamamoto S. Yoshida T. Miyoshi S. Shinoda S. Biometals. 1994; 7: 109-116Crossref PubMed Scopus (100) Google Scholar, 6Gehring A.M. DeMoll E. Fetherstone J.D. Mori I. Mayhew G.F. Blattner F.R. Walsh C.T. Chem. Biol. 1998; 5: 573-586Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 7Adilakshmi T. Ayling P.D. Ratledge C. J. Bacteriol. 2000; 182: 264-271Crossref PubMed Scopus (35) Google Scholar). The first evidence for a bacterial salicylate biosynthetic pathway was obtained in Mycobacterium smegmatis, which incorporates shikimate into salicylate and the siderophore mycobactin derived from salicylate (8Hudson A.T. Bentley R. Biochemistry. 1970; 9: 3984-3987Crossref PubMed Scopus (27) Google Scholar, 9Hudson A.T. Bentley R. Tetrahedron Lett. 1970; 24: 2077-2080Crossref Scopus (7) Google Scholar, 10Marshall B.J. Ratledge C. Biochim. Biophys. Acta. 1972; 264: 106-116Crossref PubMed Scopus (39) Google Scholar). Because isochorismate decomposes slowly into salicylate and 3-carboxylphenylpyruvate at pH 7 and room temperature, in the absence of enzymes, isochorismate was proposed to be the immediate precursor of salicylate (11Young I.G. Batterham T.J. Gibson F. Biochim. Biophys. Acta. 1969; 177: 389-400Crossref PubMed Scopus (63) Google Scholar). It was then shown that crude cell extracts of M. smegmatis convert chorismate and isochorismate to salicylate (Fig.1), by the successive action of isochorismate synthase (ICS)1 and IPL (10Marshall B.J. Ratledge C. Biochim. Biophys. Acta. 1972; 264: 106-116Crossref PubMed Scopus (39) Google Scholar, 12Marshall B.J. Ratledge C. Biochim. Biophys. Acta. 1971; 230: 643-645Crossref PubMed Scopus (14) Google Scholar). Whereas ICS has been purified from Escherichia coli (13Daruwala R. Bhattacharyya D.K. Kwon O. Meganathan R. J. Bacteriol. 1997; 179: 3133-3138Crossref PubMed Scopus (41) Google Scholar,14Dahm C. Müller R. Schulte G. Schmidt K. Leistner E. Biochim. Biophys. Acta. 1998; 1425: 377-386Crossref PubMed Scopus (30) Google Scholar), from Bacillus subtilis (15Rowland B.M. Taber H.W. J. Bacteriol. 1996; 178: 854-861Crossref PubMed Google Scholar), and recently also from a plant (16van Tegelen L.J. Moreno P.R. Croes A.F. Verpoorte R. Wullems G.J. Plant Physiol. 1999; 119: 705-712Crossref PubMed Scopus (47) Google Scholar), IPL has not yet been characterized as a pure enzyme from any organism. In the ubiquitous bacterium and opportunistic human pathogenPseudomonas aeruginosa, as in other organisms, chorismate is a precursor of aromatic amino acids, ubiquinone and folate (Fig. 1). In addition, phenazine compounds such as pyocyanin (17Essar D.W. Eberly L. Hadero A. Crawford I.P. J. Bacteriol. 1990; 172: 884-900Crossref PubMed Scopus (663) Google Scholar, 18Chin-A-Woeng T.F.C. Thomas-Oates J.E. Lugtenberg B.J.J. Bloemberg G.V. Mol. Plant Microbe Interact. 2001; 14: 1006-1015Crossref PubMed Scopus (142) Google Scholar) and the siderophore pyochelin (19Cox C.D. Rinehart K.Jr. Moore M.L. Cook J.Jr. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4256-4260Crossref PubMed Scopus (302) Google Scholar) are produced by pathways branching off from chorismate (Fig. 1). Salicylate is a biosynthetic precursor of pyochelin in P. aeruginosa (1Ankenbauer R.G. Cox C.D. J. Bacteriol. 1988; 170: 5364-5367Crossref PubMed Google Scholar, 20Ankenbauer R.G. Toyokuni T. Staley A. Rinehart K.L. Cox C.D. J. Bacteriol. 1988; 170: 5344-5351Crossref PubMed Google Scholar). Genetic evidence indicates that the pchA and pchB genes encode ICS and IPL, respectively, in this bacterium (2Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (150) Google Scholar). These genes are part of the pchDCBA operon, which, together with thepchEFGHI operon, code for the enzymes of the pyochelin pathway (21Serino L. Reimmann C. Visca P. Beyeler M. Della Chiesa V. Haas D. J. Bacteriol. 1997; 179: 248-257Crossref PubMed Google Scholar, 22Reimmann C. Serino L. Beyeler M. Haas D. Microbiology. 1998; 144: 3135-3148Crossref PubMed Scopus (104) Google Scholar, 23Quadri L.E. Keating T.A. Patel H.M. Walsh C.T. Biochemistry. 1999; 38: 14941-14954Crossref PubMed Scopus (116) Google Scholar, 24Reimmann C. Patel H.M. Serino L. Barone M. Walsh C.T. Haas D. J. Bacteriol. 2001; 183: 813-820Crossref PubMed Scopus (90) Google Scholar). The deduced protein products of the P. aeruginosa pchB gene and its Pseudomonas fluorescens homolog pmsB both show approximately 20% sequence identity with CMs of the AroQ family (25Xia T. Song J. Zhao G. Aldrich H. Jensen R.A. J. Bacteriol. 1993; 175: 4729-4737Crossref PubMed Google Scholar, 26Gu W. Williams D.S. Aldrich H.C. Xie G. Gabriel D.W. Jensen R.A. Microb. Comp. Genomics. 1997; 2: 141-158Crossref PubMed Scopus (24) Google Scholar, 27MacBeath G. Kast P. Hilvert D. Biochemistry. 1998; 37: 10062-10073Crossref PubMed Scopus (79) Google Scholar, 28Mercado-Blanco J. van der Drift K.M. Olsson P.E. Thomas-Oates J.E. van Loon L.C. Bakker P.A. J. Bacteriol. 2001; 183: 1909-1920Crossref PubMed Scopus (122) Google Scholar); the significance of this low level similarity is unclear. CM catalyzes a Claisen rearrangement of chorismate, leading to prephenate, a common precursor in phenylalanine and tyrosine biosynthesis (Fig.1). P. aeruginosa has at least two CM activities (29Patel N. Pierson D.L. Jensen R.A. J. Biol. Chem. 1977; 252: 5839-5846Abstract Full Text PDF PubMed Google Scholar, 30Zhao G. Xia T. Fischer R.S. Jensen R.A. J. Biol. Chem. 1992; 267: 2487-2493Abstract Full Text PDF PubMed Google Scholar, 31Calhoun D.H. Bonner C.A., Gu, W. Xie G. Jensen R.A. Genome Biol. 2001; 2: 0030.1-0030.16Crossref Google Scholar). The first of these is due to the bifunctional P-protein (AroQ·PheA) consisting of a CM domain (AroQ) and a prephenate dehydratase domain, which produces phenylpyruvate, a phenylalanine precursor (Fig.1). The second CM is a monofunctional, periplasmic enzyme (*AroQ). The aims of the present work are to characterize the properties of purified PchB from P. aeruginosa and to investigate its relationship to CM of the AroQ class. The endo-oxabicyclic diacid transition state analog inhibitor of CM and sodium isochorismate (used as a reference) were generous gifts from Drs. P. Bartlett (University of California, Berkeley, CA) and E. W. Leistner (University of Cologne, Cologne, Germany), respectively. Prephenic acid and phenylpyruvic acid were obtained from Sigma (Fluka Chemie AG, Switzerland). Carbenicillin was a gift from Glaxo-SmithKline. Chorismic acid was isolated from the CM-deficient Klebsiella pneumoniae strain 62-1 (ATCC 25306; Phe−Tyr− Trp−; Ref. 32Gibson F. Biochem. J. 1964; 90: 256-261Crossref PubMed Scopus (98) Google Scholar), purified as the free acid according to the method of Grisostomi et al. (33Grisostomi C. Kast P. Pulido R. Huynh J. Hilvert D. Bioorg. Chem. 1997; 25: 297-305Crossref Scopus (42) Google Scholar) and stored at −80 °C. The purity of chorismic acid was 98% as determined by UV spectroscopy using ε274 nm = 2,630m−1 cm−1 (34Heide E. Morrison J.F. Biochemistry. 1978; 17: 1573-1580Crossref PubMed Scopus (40) Google Scholar) and 95.3% by analytical C-18 reverse phase HPLC at 280 nm. Prephenic acid, phenylpyruvic acid, and p-hydroxybenzoic acid were present at 0.6, 1.4, and 0.06%, respectively, as judged by HPLC. Isochorismate was isolated from K. pneumoniae 62-1 harboring theentC plasmid pKS3-02 (35Schmidt K. Leistner E. Biotechnol. Bioeng. 1995; 45: 285-291Crossref PubMed Scopus (19) Google Scholar), which was grown in medium A containing kanamycin (60 μg/ml), and transferred to 2-liter production medium BN (11Young I.G. Batterham T.J. Gibson F. Biochim. Biophys. Acta. 1969; 177: 389-400Crossref PubMed Scopus (63) Google Scholar) containing 0.1 mm each of tyrosine, phenylalanine, and tryptophan plus 0.3 mmisopropyl-β-d-thio-galactopyranoside (IPTG) (35Schmidt K. Leistner E. Biotechnol. Bioeng. 1995; 45: 285-291Crossref PubMed Scopus (19) Google Scholar). After precipitation with 2 m barium acetate, a 1:2 mixture of isochorismate and chorismate (600 mg from 2 liters of culture) was obtained, of which 50-mg portions were purified on a Nucleosil 120 C-18 column (10 × 250 mm) in aqueous 0.27% formic acid, pH 2.5. Elution at 4.1 ml/min was carried out by a methanol (%, v/v) step gradient: 10 min, 0%; 10–16 min, 0–19%; 16–41 min, 19%; 41–51 min, 19–28.5%; 51–58 min, 28.5%; 58–63 min, 28.5–95%; 63–73 min, 95% (35Schmidt K. Leistner E. Biotechnol. Bioeng. 1995; 45: 285-291Crossref PubMed Scopus (19) Google Scholar). 2E. W. Leistner, personal communication. Isochorismic acid and chorismic acid were eluted with 19% methanol after 30 min and with 28.5% methanol after 48 min, respectively, neutralized with NaOH, and freeze-dried. Isochorismate (ε278 nm = 8,300 m−1cm−1; Ref. 36Rusnak F. Liu J. Quinn M. Berchtold G.A. Walsh C.T. Biochemistry. 1990; 29: 1425-1435Crossref PubMed Scopus (82) Google Scholar) preparations adjusted to 200 μm contained 65 mm sodium formate, 43 μm sodium prephenate, and 20 μm sodium phenylpyruvate. No contamination of the isochorismate preparations with chorismate or with salicylate could be detected. At an isochorismate concentration giving half-maximal velocity (12.5 μm), the addition of 20 μm sodium prephenate, 8 μmsodium phenylpyruvate, or 26 mm sodium formate (i.e. a 10-fold excess over the contamination levels present in our isochorismate preparations) had no effect on IPL activity. An analytical detection method at pH 7.0 was worked out for isochorismate, using a Nucleosil C-18 column (4 × 250 mm). Elution at a flow rate of 1 ml/min was carried out with a 0–65% (v/v) linear gradient of acetonitrile in 10 mm potassium phosphate buffer, pH 7.0, containing 5 mm ion pair reagent tetrabutylammonium hydrogen sulfate (Fluka), using a Hewlett-Packard 1050 system equipped with a diode array detector. p-Hydroxybenzoate (10.2 min), prephenate (10.9 min), chorismate (11.7 min), isochorismate (12.4 min), phenylpyruvate (15.6 min), and salicylate (16.4 min) were eluted at the retention times indicated and quantitated at λmax with a diode array UV detector. Routine procedures were used for the isolation of DNA and for cloning experiments (37Del Sal G. Manfioletti G. Schneider C. Nucleic Acids Res. 1988; 16: 9878Crossref PubMed Scopus (230) Google Scholar, 38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). K. pneumoniae and E. colistrains were transformed with plasmid DNA by electroporation (39Farinha M.A. Kropinski A.M. FEMS Microbiol. Lett. 1990; 77: 221-226Google Scholar). Plasmid pME6152, containing pchB under the tacpromoter, was constructed by cloning the 0.75-kb pchB XhoI-PstI fragment of pME3368 (2Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (150) Google Scholar) in pMMB67EH (40Fürste J.P. Pansegrau W. Frank R. Blöcker H. Scholz P. Bagdasarian M. Lanka E. Gene (Amst.). 1986; 48: 119-131Crossref PubMed Scopus (813) Google Scholar). A 0.75-kb BamHI/HindIII fragment encodingpchB(I88T) from mutated pME6152 (see below) was subcloned into pUK21 (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and pMMB67EH, resulting in pME6169 and pME6179, respectively. The nucleotide sequence of the pchB(I88T) gene on pME6179 was determined by using the Thermo Sequenase II dye terminator cycle sequencing kit (Amersham Biosciences) and the ABI Prism™ 373 sequencer. P. aeruginosaADD1976/pME3324 (a T7 promoter expression vector with pchB; Ref. 2Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (150) Google Scholar) was grown in 250 ml of nutrient yeast broth (2Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (150) Google Scholar) containing carbenicillin (250 μg/ml) in 1-liter flasks with shaking at 37 °C. At A600 nm = 0.8, the chromosomal T7 RNA polymerase gene was induced for 1 h by the addition of IPTG at 1 mm final concentration. Rifampicin was then added at 200 μg/ml, and the incubation was continued for 2 h. All subsequent operations were carried out at 4 °C. Cells from three flasks were harvested by centrifugation at 8,000 × g for 10 min and washed twice in 50 mm potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol and 1 mm dithiothreitol (DTT) (buffer A). Cells (wet weight of 3 g), which could be stored at −20 °C, were broken in 15 ml of buffer A by sonication for 5 × 30 s. Cell debris was removed by centrifugation at 10,000 × g for 30 min; the resulting crude extract containing 8–10 mg/ml protein was applied to a 1.6 × 20-cm DEAE-Sepharose CL-6B column (Amersham Biosciences) equilibrated with buffer A. The enzyme was eluted with 300 ml of buffer A at a flow rate of 1 ml/min. The fractions containing PchB, detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 10–20% gradient gels stained with Coomassie Blue, were combined (100 ml) and loaded onto a column of phenyl-Sepharose CL-4B (1.6 × 10 cm) previously equilibrated with 200 ml of buffer A. The enzyme was eluted as a broad peak with 150 ml of buffer A. Fractions containing PchB were pooled (80 ml), and a solution of 0.4% (w/v)n-octyl-β-glucopyranoside (Calbiochem) was slowly added by stirring to a final concentration of 0.1% (w/v), to avoid protein precipitation during the concentration steps. This fraction was concentrated to 2 ml by ultrafiltration through an Amicon YM3 membrane in a stirred cell, followed by further concentration with an Amicon Centriplus C-10 concentrator (cut-off: 10,000 Da). The concentrated fraction was chromatographed on a 1.6 × 70-cm column of Bio-Gel P100 polyacrylamide gel (Bio-Rad) in buffer A at a flow rate of 6 ml/h. PchB-containing fractions were pooled, aliquoted, and stored at −80 °C. The protein concentration was determined by the method of Bradford (41Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) using a commercial reagent (Bio-Rad) and bovine serum albumin as the standard. The subunit molecular mass of PchB was estimated by SDS-PAGE (42Lämmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3025) Google Scholar) in 10–20% gradient gels (Ready Gel, Bio-Rad), with standards of the Low Molecular Weight Calibration Kit (Amersham Biosciences). The molecular mass of the native PchB was estimated by gel filtration chromatography on Sephadex G-150 (1.6 × 70 cm, 0.1 ml/min), Bio-Gel P100 (1.6 × 70 cm, 0.1 ml/min), and FPLC Superose 12 HR (10/30, 0.4 ml/min) columns in buffer A, with ribonuclease A (13.7 kDa), lysozyme (14.6 kDa), proteinase K (28.8 kDa), pepsin (34.5 kDa), protein A (42 kDa), ovalbumin (43 kDa), and bovine serum albumin (67 kDa) as markers. The elution volumes were plotted against the logarithm of molecular masses for the standards, and the linear regression curve was used to estimate the apparent molecular mass of PchB. In addition, the molecular mass of native PchB was estimated from PAGE in nondenaturing gels of 7.5, 10, 12, 15, and 20% polyacrylamide, with the Low Molecular Weight Calibration Kit (Amersham Biosciences) as a standard, by Ferguson plot analysis (43Tietz D. Chrambach A. Anal. Biochem. 1987; 161: 395-411Crossref PubMed Scopus (48) Google Scholar). The slopes obtained from plots of the logarithm of relative mobility versuspolyacrylamide concentration were plotted against the molecular weight of the standard proteins. The N-terminal sequence of PchB was determined by Dr. P. James (ETH, Zürich, Switzerland) on an Applied Biosystems Peptide Sequencer model 473A, using the Edman degradation procedure. Samples of 50 μl of purified PchB (0.54 μg/μl) in buffer A were treated with 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) to a final concentration of 0.5 mm. The cross-linker was prepared immediately before use as a 5 mm solution in 50 mm potassium phosphate, pH 7.5, containing 0.75m NaCl. The samples were incubated with constant agitation at room temperature for 30 min. The reaction was quenched by addition of 40 mm Tris-HCl buffer, pH 7.5. Each sample was split into two aliquots, treated with the solubilizing solution (final concentrations: 1.2% (w/v) SDS, 10% (w/v) sucrose, 10 mmTris-HCl, pH 7.5) with or without 40 mm DTT to cleave the cross-links, incubated at 50 °C for 3 min, and subjected to SDS-PAGE on a 10–20% polyacrylamide gradient. Standard proteins (Low and High Molecular Weight Kits, Amersham Biosciences) and the PchB control were not exposed to DTSSP, but were otherwise treated in the same way. IPL activity of PchB was determined by measuring the fluorescence of salicylate formed using an excitation wavelength of 305 nm and an emission wavelength of 440 nm in a PerkinElmer LS30 fluorimeter. Unless stated otherwise, the incubation mixture contained, in a final volume of 500 μl, 100 mmpotassium phosphate buffer, pH 7.0, 600 μm isochorismate (purified by HPLC), 10% (v/v) glycerol, 10 mmMgCl2, 1 mm DTT, and enzyme. Controls without enzyme were included routinely, to correct for nonenzymatic reactions. The reaction at 37 °C was initiated by the addition of isochorismate and terminated by the addition of 10 μl of concentrated HCl, followed by extraction with 3 ml of ethyl acetate. Blanks were obtained from nonincubated reaction mixtures. The amount of salicylate formed was determined from a standard curve obtained with 0.5–8 μmsalicylic acid (Fluka) in ethyl acetate. One IPL unit of enzyme activity is defined as the amount of enzyme converting 1 μmol of isochorismate to salicylate/min. The reaction was stopped within the first 10% of the isochorismate-to-salicylate conversion. CM activity of PchB was assayed by the method of Cotton and Gibson (44Cotton R.G.H. Gibson F. Biochim. Biophys. Acta. 1965; 100: 76-88Crossref PubMed Scopus (35) Google Scholar) as modified by Ahmad and Jensen (45Ahmad S. Jensen R.A. Mol. Biol. Evol. 1988; 5: 282-297PubMed Google Scholar). The product of the enzymatic reaction, prephenate, is converted to phenylpyruvate under acidic conditions, which is subsequently measured by its absorbance at 320 nm at basic pH. The reaction mixture contained, in a final volume of 0.2 ml, 50 mm potassium phosphate, pH 7.0, 1 mmDTT, 1 mm chorismate, and enzyme. Control reaction mixtures lacking enzyme were always included to estimate the amount of prephenate formed nonenzymatically during the incubation period. Reactions were carried out at 37 °C and terminated by the addition of 0.1 ml of 1 m HCl, followed by incubation at 37 °C for 10 min to convert the prephenate formed to phenylpyruvate. Phenylpyruvate concentrations were determined at 320 nm after the addition of 0.7 ml of 2.5 m NaOH, using ε320 nm = 17,500 m−1 cm−1 (44Cotton R.G.H. Gibson F. Biochim. Biophys. Acta. 1965; 100: 76-88Crossref PubMed Scopus (35) Google Scholar). One CM unit of enzyme activity is defined as the amount of enzyme needed for the formation of 1 μmol of prephenate (assayed as phenylpyruvate)/min. Assays were performed in triplicate and repeated three times. Initial velocity data were fitted to the equations of Hanes (Table II), Lineweaver-Burk (Fig. 3, A–C), and Dixon using Enzpack software (Biosoft).Table IIKinetic parameters of PchB for IPL and CM activities at 37 °CActivitySubstrateKmVmaxkcatKi2-aFor the determination of the inhibition mode, data were fitted to the Lineweaver-Burk equation (v−1 = KmVmax−1 [S]−1 +Vmax−1) (see Fig. 3). TheKi values were calculated from a Dixon plot (v−1 against [I]).TSAChorismateμmμmol min−1min−1μmμmIPLIsochorismate12.54.9 × 10−310622400CMChorismate1503.4 × 10−37.828Three to four concentrations of both TSA (10–200 μm) and chorismate (100 μm to 1 mm) were tested.2-a For the determination of the inhibition mode, data were fitted to the Lineweaver-Burk equation (v−1 = KmVmax−1 [S]−1 +Vmax−1) (see Fig. 3). TheKi values were calculated from a Dixon plot (v−1 against [I]). Open table in a new tab Three to four concentrations of both TSA (10–200 μm) and chorismate (100 μm to 1 mm) were tested. The CM-negative E. coli strain KA12 harboring pKIMP-UAUC (47Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar, 48Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar) with or without pME6152 (pchB+) was grown at 37 °C with shaking in 500-ml flasks containing 100 ml of minimal medium E (48Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar), amended with 0.5% (w/v) glucose, 1 mm thiamine, and 1 mm IPTG. Control cultures were grown in minimal medium E supplemented with 1 mm tyrosine and 1 mmphenylalanine. The cultures were inoculated with approximately 107 washed cells/ml, previously grown in minimal medium E supplemented with both amino acids, ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml). Cell growth was monitored by following the A600 nm. Rabbit antibodies were generated by subcutaneous injection of purified PchB (110 μg/500 μl) emulsified with 500 μl of complete Freund's adjuvant. Three booster injections with the same amount of antigen at 4-week intervals were applied. Ten days after the final injection, blood was collected and aliquots of the serum obtained were kept at −80 °C. The enzyme (typically 0.1 μg) and cell extracts were subjected to SDS-PAGE and electrotransferred to a nitrocellulose sheet (Bio-Rad) in a Trans-Blot apparatus (Bio-Rad) with a transfer buffer containing 25 mmTris base (pH 8.3), 192 mm glycine, and 20% (v/v) ethanol. The antiserum was diluted 1:1,000 with Tris-buffered saline-Tween (20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 3 mm KCl, 0.05% (w/v) Tween 20) for use in PchB immunoblots, which were treated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma). PchB was visualized by the enhanced chemiluminescence system according to the manufacturer's instructions (Amersham Biosciences). IPL was purified from the overproducing strain P. aeruginosa ADD1976/pME3324 as described under “Experimental Procedures” and summarized in Table I. In crude extract, PchB constituted ∼20% of the soluble protein. Purification by DEAE-ion exchange chromatography resulted in a 5.9-fold increase in specific activity. Hydrophobic interaction chromatography on phenyl-Sepharose and molecular sieving on Bio-Gel P100 removed some contaminants, but did not improve the specific activity (Table I). PchB protein tended to form insoluble aggregates in concentrated solution. Therefore, pooled fractions after phenyl-Sepharose chromatography were concentrated by ultrafiltration in the presence of 0.1%n-octyl-β-d-glucopyranoside, allowing a higher recovery. Used at this concentration, n-octylglucose had no effect on IPL activity. Glycerol, which was present in buffer A, did not influence IPL activity either, in concentrations of up to 30%. PchB was eluted from the Bio-Gel column in a single symmetric peak, with a purity of >98% as judged by SDS-PAGE (Fig.2, lane 1). The PchB polypeptide (calculated molecular mass of 11.5 kDa) migrated slightly ahead of the 14.4-kDa marker, α-lactalbumin (Fig. 2).Table IPurification of the PchB enzyme from the overproducing strain P. aeruginosa ADD1976/pME3324, based on IPL activityPurification stepProteinActivitySpecific activityYieldPurificationmgunitsunits/mg%-foldCrude cell extract107.4251.32.341001DEAE-Sepharose CL-6B14.6200.613.7479.85.9Phenyl-Sepharose CL-4B8.9100.811.3340.14.8Bio-Gel P1004.456.712.8922.65.5 Open table in a new tab The N-terminal amino acid sequence of the PchB polypeptide (Met-Lys-Thr-Pro-Glu-Asp-X-Thr-Gly-Leu) obtained by Edman degradation matched that predicted from the DNA sequence (2Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (150) Google Scholar) after processing of the first methionine. Mobility of PchB in PAGE under nondenaturing conditions was similar to that of carbonic anhydrase (28.7 kDa), and a Ferguson plot indicated an apparent molecular mass of 30 ± 2 kDa. Analytical size exclusion chromatography on Sephadex G-150, Superose 12 HR, and Bio-Gel P100 gave values of ∼32, 31 ± 1, and 34 ± 2 kDa, respectively, for native PchB. PchB was cross-linked with the homobifunctional, water-soluble agent DTSSP and treated with the solubilizing buffer as described under “Experimental Procedures.” After SDS-PAGE, a substantial fraction of PchB was found to migrate as a covalently linked dimer (Fig. 2, lane 2). Larger forms corresponding probably to trimers and tetramers were also seen with much lower intensities (Fig. 2, lane 2). Increasing the cross-linker concentration to 2 mm appeared to favor the dimeric form (data not shown). Treatment of cross-linked PchB with DTT converted these forms back to the monomeric subunit (data not shown). The fact that the band corresponding to the dimer was dominant after cross-linking can be interpreted in favor of a dimeric structure of native PchB. The apparent molecular mass obtained by molecular sieving would also be compatible with a trimeric PchB structure. However, CM ofMethanococcus jannaschii (27MacBeath G. Kast P. Hilvert D. Biochemistry. 1998; 37: 10062-10073Crossref PubMed Scopus (79) Google Scholar), a dimeric protein whose 99-amino acid sequence is related to that of PchB (see below), showed an apparent molecular mass of 29.4 kDa in gel filtration experiments. This value is also higher than that expected for a dimeric form and was assumed to resu" @default.
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