γ-Glutamylputrescine Synthetase in the Putrescine Utilization Pathway of Escherichia coli K-12
2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês
10.1074/jbc.m800133200
ISSN1083-351X
AutoresShin Kurihara, Shinpei Oda, Yuichi Tsuboi, Hyeon Guk Kim, Mayu Oshida, Hidehiko Kumagai, Hideyuki Suzuki,
Tópico(s)Biopolymer Synthesis and Applications
ResumoGlutamate-putrescine ligase (γ-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the γ-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving γ-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent γ-glutamylation of putrescine. The Km values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mm, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without γ-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway. Glutamate-putrescine ligase (γ-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the γ-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving γ-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent γ-glutamylation of putrescine. The Km values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mm, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without γ-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway. γ-Glutamyl linkage is an amide linkage between the γ-position carboxyl group of glutamate and an amino group of various compounds. Compounds that have a γ-glutamyl linkage are called γ-glutamyl compounds, which are widely found in both prokaryotic and eukaryotic cells. For example, the peptidoglycan of Escherichia coli has a γ-glutamyl linkage between d-glutamate and meso-2,6-diaminopimelic acid (1Park, J. T. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., and Umbarger, H. E., eds) 2nd Ed., pp. 49–57, American Society for Microbiology, Washington, DCGoogle Scholar), the virulence of Bacillus anthracis is dependent on a capsule made of poly-γ-glutamic acid (2Candela T. Fouet A. Mol. Microbiol. 2006; 60: 1091-1098Crossref PubMed Scopus (264) Google Scholar), theanine (γ-glutamylethylamide) is a major “umami” component of Japanese green tea (3Suzuki H. Izuka S. Minami H. Miyakawa N. Ishihara S. Kumagai H. Appl. Environ. Microbiol. 2003; 69: 6399-6404Crossref PubMed Scopus (52) Google Scholar), and glutathione (γ-glutamylcysteinylglycine) is a very important antioxidant (4Mannervik, B., Carlberg, I., and Larson, K. (1989) Coenzymes and Cofactors, pp. 475–516, Wiley-Interscience, New YorkGoogle Scholar) in living cells.Putrescine is one of the polyamines that are found in a wide range of organisms from bacteria to plants and animals; are critical for cell proliferation, differentiation, and transformation; and are involved in DNA, RNA, and protein synthesis as well as in stabilizing membrane and cytoskeletal structures (5Tabor C.W. Tabor H. Microbiol. Rev. 1985; 49: 81-99Crossref PubMed Google Scholar, 6Pegg A.E. Biochem. J. 1986; 234: 249-262Crossref PubMed Scopus (1426) Google Scholar). An increased concentration of polyamine is observed in cancer cells (7Linsalata M. Caruso M.G. Leo S. Guerra V. D'Attoma B. Di Leo A. Anticancer Res. 2002; 22: 2465-2469PubMed Google Scholar), there is a significantly elevated concentration (more than millimolar) of putrescine in plants under various stress conditions, and its concentration is very high (estimated to be over 30 mm) in E. coli cells (8Igarashi K. Kashiwagi K. Biochem. Biophys. Res. Commun. 2000; 271: 559-564Crossref PubMed Scopus (718) Google Scholar).We previously reported (9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) that putrescine imported from medium by PuuP, a putrescine importer, is degraded to succinic semialdehyde, a precursor of succinate, via γ-glutamyl intermediates by products of the puu gene cluster (Fig. 1). In this metabolic pathway, which we named the Puu pathway (Fig. 2), putrescine is first γ-glutamylated to γ-Glu-Put 4The abbreviations used are: γ-Glu-Put, γ-glutamylputrescine; GS, glutamine synthetase; GABA, γ-aminobutyrate; γ-Glu-GABA, γ-glutamyl-γ-aminobutyrate; RT, reverse transcription; HPLC, high pressure liquid chromatography; Put, putrescine. 4The abbreviations used are: γ-Glu-Put, γ-glutamylputrescine; GS, glutamine synthetase; GABA, γ-aminobutyrate; γ-Glu-GABA, γ-glutamyl-γ-aminobutyrate; RT, reverse transcription; HPLC, high pressure liquid chromatography; Put, putrescine. by PuuA (glutamate-putrescine ligase, γ-Glu-Put synthetase, EC 6.3.1.11). Secondly γ-Glu-Put is oxidized to γ-Glu-γ-butyraldehyde by PuuB (γ-Glu-Put oxidase) and further oxidized to γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) by PuuC (γ-Glu-γ-butyraldehyde dehydrogenase). Then γ-Glu-GABA is hydrolyzed to glutamate and GABA by PuuD (γ-Glu-GABA hydrolase) (10Kurihara S. Oda S. Kumagai H. Suzuki H. FEMS Microbiol. Lett. 2006; 256: 318-323Crossref PubMed Scopus (24) Google Scholar), and then GABA is deaminated to succinic semialdehyde by PuuE (GABA:α-ketoglutarate aminotransferase). PuuA catalyzes the first reaction of this Puu pathway as shown below (Scheme 1). Glutamate+Putrescine+ATP→γ-Glutamylputrescine +H2O+ADP+PiScheme 1 Putrescine has been reported to be degraded without γ-glutamylation (11Shaibe E. Metzer E. Halpern Y.S. J. Bacteriol. 1985; 163: 938-942Crossref PubMed Google Scholar, 12Samsonova N.N. Smirnov S.V. Novikova A.E. Ptitsyn L.R. FEBS Lett. 2005; 579: 4107-4112Crossref PubMed Scopus (35) Google Scholar, 13Samsonova N.N. Smirnov S.V. Altman I.B. Ptitsyn L.R. BMC Microbiol. 2003; 3: 2-11Crossref PubMed Scopus (48) Google Scholar)(not γ-glutamylated) to γ-aminobutyraldehyde by YgjG (putrescine:α-ketoglutarate aminotransferase) (13Samsonova N.N. Smirnov S.V. Altman I.B. Ptitsyn L.R. BMC Microbiol. 2003; 3: 2-11Crossref PubMed Scopus (48) Google Scholar) and subsequently converted to GABA by YdcW (γ-aminobutyraldehyde dehydrogenase) (12Samsonova N.N. Smirnov S.V. Novikova A.E. Ptitsyn L.R. FEBS Lett. 2005; 579: 4107-4112Crossref PubMed Scopus (35) Google Scholar) (Fig. 2); however, γ-aminobutyraldehyde, the intermediate of this pathway, is unstable (11Shaibe E. Metzer E. Halpern Y.S. J. Bacteriol. 1985; 163: 938-942Crossref PubMed Google Scholar) and is non-enzymatically cyclized to Δ1-pyrroline (Fig. 2). γ-Glutamylation is thought to stabilize the intermediate, inhibiting the spontaneous cyclization reaction of γ-aminobutyraldehyde to Δ1-pyrroline (9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) (Fig. 2).FIGURE 2Schematic presentation of putrescine degradation pathway of E. coli. PuuA, PuuB, PuuC, and PuuD comprise the Puu pathway to degrade putrescine to GABA via γ-glutamylated intermediates. YgjG and YdcW comprise the traditional pathway to degrade putrescine to GABA without γ-glutamylation. It was reported that PuuR (10Kurihara S. Oda S. Kumagai H. Suzuki H. FEMS Microbiol. Lett. 2006; 256: 318-323Crossref PubMed Scopus (24) Google Scholar), ArcA, and FNR (27Partridge J.D. Scott C. Tang Y. Poole R.K. Green J. J. Biol. Chem. 2006; 281: 27806-27815Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) are transcriptional repressors of puuA and puuD. γ-Glu-γ-aminobutyraldehyde, γ-glutamyl-γ-aminobutyraldehyde; α-KG, α-ketoglutarate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PuuA was initially annotated as the putative glutamine synthetase (GS) by computer analysis based on the amino acid sequence. GS (14Stadtman E.R. J. Biol. Chem. 2001; 276: 44357-44364Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) catalyzes the condensing reaction of glutamate and ammonia with the aid of ATP (Scheme 2). Glutamate+Ammonia +ATP→Glutamine+H2O+ADP +PiScheme 2 Schemes 1 and 2 are very similar because both reactions are γ-glutamylation, the condensing reaction yielding an amide linkage between the γ-carboxyl group of glutamate and the amino group of putrescine or ammonia using ATP.In this study, we report that PuuA is an important enzyme that catalyzes the first step of the Puu pathway and is regulated by a complicated regulation system. We also discuss the similarities between GS and PuuA.MATERIALS AND METHODSStrain and Plasmid ConstructionThe strains, plasmids, and oligonucleotides used in this study are listed in Table 1. The strains were derivatives of E. coli K-12. Strain SH639 (15Suzuki H. Kumagai H. Echigo T. Tochikura T. J. Bacteriol. 1989; 171: 5169-5172Crossref PubMed Google Scholar) has a deletion of the ggt gene. P1 transduction, DNA manipulation, and transformation were performed by the standard methods (16Miller, J. H. (1992) A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Bacteria, pp. 263–274 and 437, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar, 17Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., pp. 1.1–2.110, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar). A DNA fragment of Kohara clone 257 (18Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1070) Google Scholar) containing the puu gene cluster was used to make plasmids. The cloned regions of DNA on plasmids and the deleted regions of DNA are summarized in Fig. 1. In strain SK212, disruption of the puuR gene was carried out as follows. The 4.5-kb EcoRV-EcoRV fragment, including puuADR, was isolated from Kohara phage 257 and cloned between the XmnI site and the blunt-ended ScaI site of pACYC184 to obtain pKHG3. The 220-bp region between EcoRI and XmnI sites was replaced with the 1.2-kb HincII kanamycin resistance cassette of pUC4K. The plasmid was linearized using SalI. Homologous recombination was performed using this linearized plasmid to delete residues 65–137 of a putative 185-residue PuuR protein (Fig. 1). In SK310, the puuA gene was disrupted according to the method of Datsenko and Wanner (19Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar) using oligonucleotides puuA-1 and puuA-2. This strain deleted the predicted ATP binding motif of PuuA (Figs. 1 and 8). In SO58, disruptions of puuADR and puuCBE were performed by the modified method of Datsenko and Wanner (19Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar) as follows. To disrupt puuCBE, the DNA fragment of Kohara clone 257 (18Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1070) Google Scholar), containing the puuCBE gene region, was digested with NarI and HincII and cloned into pUC19 digested with NarI and HincII to obtain pSK169. The FRT-kan+-FRT fragment was amplified by PCR using oligonucleotides pKD13-1 and pKD13-4 as primers and plasmid pKD13 as a template with KOD-plus DNA polymerase (Toyobo, Osaka, Japan). The PCR product was ligated with the 3.9-kb DraIII (blunt-ended with a Blunting kit (Takara, Kyoto, Japan)) and EcoRV fragment of pSK169, which deleted all of the puuB and most of the puuC and puuE genes (Fig. 2). The obtained plasmid was cleaved with HindIII and AatII, and the 3.0-kb fragment was used to transform TK251 by electroporation at 30 °C. Kanamycin-resistant transformant SK231 was obtained. To disrupt puuADR, the FRT-cat+-FRT fragment was amplified by PCR using oligonucleotides pKD13-1 and pKD3-2 as primers and plasmid pKD3 as a template with KOD-plus DNA polymerase. The PCR product was ligated with the 4.7-kb XmnI and HpaI fragment of pKHG3, which deleted all of the puuAD and most of the puuR genes. The obtained plasmid was cleaved with SacII and NruI, and the 2.8-kb fragment was used to transform SK231 by electroporation. Chloramphenicol-resistant transformant SO56 was obtained. Then SO58 was made by P1 transduction of the ΔpuuADR::(FRT-cat+-FRT) ΔpuuCBE::(FRT-kan+-FRT) alleles from SO56 to SH639. To construct pKHG8, a 2.0-kbp SmaI and HpaI fragment of pKHG3 was ligated with pBR322 digested with EcoRV and NruI. pSO105, which carries puuAH282N, was constructed with the QuikChange technique (Stratagene) using oligonucleotides puuA-3 and puuA-4 as primers and pSO97 as a template but using KOD-plus DNA polymerase (Toyobo). pSK324, which carries puuAR357Q, was similarly constructed using oligonucleotides puuA-5 and puuA-6.TABLE 1Strains, plasmids, and oligonucleotides used in this studyStrain, plasmid, or oligonucleotideCharacteristic or sequenceSource or Ref.Strain KP7600W3110 but F- lacIq lacZΔM15 galK2 galT22 lambda- in (rrnD-rrnE)1T. Miki JD22571KP7600 but ΔydcW::mini-Tn10kanT. Miki JD24090KP7600 but ΔygjG::mini-Tn10kanT. Miki MG1655F- prototrophicC. A. Gross SH639F- Δggt-215Suzuki H. Kumagai H. Echigo T. Tochikura T. J. Bacteriol. 1989; 171: 5169-5172Crossref PubMed Google Scholar SK212SH639 but ΔpuuR::kan+This study SK231pKD46/F- ΔpuuCBE::(FRT-kan+-FRT)This study SK247SH639 but ΔpuuDRCBE::FRT9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar SK293pBR322/SK247This study SK308pBelobac11/SH639This study SK310SH639 but ΔpuuA-2::FRTThis study SK326pSK324/YG110This study SK327pSK325/YG110This study SK350SH639 but ΔydcW::mini-Tn10kanThis study; SH639 X P1(JD22571) SK351SH639 but ΔpuuA-2::FRT ΔydcW::mini-Tn10kanThis study; SK310 X P1(JD22571) SK352SH639 but ΔygjG::mini-Tn10kanThis study; SH639 X P1(JD24090) SK353SH639 but ΔpuuA-2::FRT ΔygjG::mini-Tn10kanThis study; SH310 X P1(JD24090) SO56F- ΔpuuADR::(FRT-cat+-FRT) ΔpuuCBE::(FRT-kan+-FRT)This study SO58SH639 but ΔpuuADR::(FRT-cat+-FRT) ΔpuuCBE::(FRT-kan+-FRT)This study SO62pBBR322/SO58This study SO63pKHG8/SO58This study SO97pSO97/YG110This study SO106pSO105/YG110This study SO115pSO82/SK310This study SO116pBelobac11/SK310This study TK251pKD46/MG1655Laboratory stock YG110F- hsdS gal (rB-mB-) ΔtyrR::kan+ (DE3)Laboratory stock Kohara phage clone 25718Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1070) Google ScholarPlasmid pACYC184p15A replicon cat+ tet+New England Biolabs pBelobac11Mini-F replicon cat+New England Biolabs pBR322ColE1 replicon rop+ bla+ tet+New England Biolabs pKD3oriRγ bla+ FRT-cat+-FRT19Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar pKD13oriRγ bla+ FRT-kan+-FRT19Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar pKD46oriR101 replicon repA101ts araC+ gam+-bet+-exo+ (araBP) bla+19Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar pKHG3p15A replicon rop+ tet+ puuA+ puuD+ puuR+This study pKHG8ColE1 replicon rop+ bla+ puuA+This study pSK169ColE1 replicon bla+ puuC+ puuB+ puuE+This study pSK324ColE1 replicon bla+ puuAR357Q (T7P)This study pSK325ColE1 replicon bla+ puuAH282N/R357Q (T7P)This study pSO82Mini-F replicon cat+ puuA+This study pSO97ColE1 replicon bla+ puuA+ (T7P)This study pSO105ColE1 replicon bla+ puuAH282N (T7P)This study pUC19ColE1 replicon bla+New England Biolabs pUC4KColE1 replicon bla+ lacZα::kan+GE HealthcareOligonucleotide pKD3-25′-CATATGAATATCCTCCTTA-3′ pKD13-15′-GTGTAGGCTGGAGCTGCTTC-3′ pKD13-45′-ATTCCGGGGATCCGTCGACC-3′ puuA-15′-GCCTTCCGCGTCAGAAAGCACGTTCTCGCCACGATTATTTTGCATGTGTAGGCTGGAGCTGCTTC-3′ puuA-25′-CGTCTGATGGCAGAAAAGCATAAGATGCACGCCACTTTTATGGCGATTCCGGGGATCCGTCGACC-3′ puuA-35′-GCATACTGATATGGATGTTCATTCCGCTGCCCGC-3′ puuA-45′-GCGGGCAGCGGAATGAACATCCATATCAGTATGC-3′ puuA-55′-GCCGCACGGAATCTGCAGGGCGACGGTG-3′ puuA-65′-CACCGTCGCCCTGCAGATTCCGTGCGGC-3′ puuA-RT15′-AAGGAACCGAGAACAGGAACAC-3′ puuA-RT25′-GCAATGGATATTCTGGGCAAC-3′ gapA-RT15′-TGAAAGGCTTCTGGGCTACACT-3′ gapA-RT25′-CGAAGTTGTCGTTTCAGAGCGATACC-3′ Open table in a new tab FIGURE 8Alignment of PuuA and GS of E. coli K-12. Identical and similar amino acid residues are indicated by black and gray shading, respectively. Under the alignment, the amino acid residues involved in the n1, n2, ammonium ion, glutamate, and ATP binding sites in GS are indicated. Above the alignment, two mutated amino acid residues in PuuA and a putative ATP binding motif deduced in PROSITE are indicated. Gaps are indicated by dashes. Alignment was performed by the needle program of EMBOSS-Align, and shading was performed using the BOXSHADE program.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Media and Growth of BacteriaIn all experiments, except when studying the influence of the overexpression of native PuuA on protein purification, strains were grown at 37 °C with reciprocal shaking at 140 rpm in 60 ml of medium in a 300-ml Erlenmeyer flask. M9-tryptone (M9 minimal medium (16Miller, J. H. (1992) A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Bacteria, pp. 263–274 and 437, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar) except that 1% Bacto tryptone was used instead of 0.2% glucose) was used in the analysis of the intracellular amino acid and polyamine profiles. In the study to determine whether E. coli can grow using putrescine as the sole source of nitrogen, W-Glc-Put medium (W salts minimal medium (20Smith G.R. Halpern Y.S. Magasanik B. J. Biol. Chem. 1971; 246: 3320-3329Abstract Full Text PDF PubMed Google Scholar) containing 0.4% glucose as the sole carbon source and 0.2% putrescine as the sole nitrogen source) was used. To determine whether E. coli can grow using putrescine as the sole source of carbon, M9-Put-AS medium (M9 minimal medium (16Miller, J. H. (1992) A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Bacteria, pp. 263–274 and 437, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar) except that 0.4% putrescine and 0.4% ammonium sulfate were used instead of 0.2% glucose and 0.2% ammonium chloride, respectively) was used. In growth experiments in carbon- and nitrogen-limited medium, strains were precultured on an LB plate at 37 °C, streaked on a nutrient-limited plate, and incubated at 20 °C. To overexpress mutagenized PuuA, strains were grown in 60 ml of LB broth containing 100 μg/ml ampicillin at 37 °C with shaking at 140 rpm in a 300-ml Erlenmeyer flask. To overexpress native PuuA for protein purification, strains were grown in 200 ml of LB broth containing 100 μg/ml ampicillin at 37 °C with shaking at 140 rpm in a 1-liter Erlenmeyer flask. γ-Glu-Put was enzymatically synthesized and purified as described previously (9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).Analysis of Amino Acids and PolyaminesAmino acids and polyamines in the samples were measured using an HPLC system (model LC-20AD; Shimadzu, Kyoto, Japan) equipped with a Shim-pack Amino-Na column (Shimadzu) with gradient elution at 60 °C at a flow rate of 0.6 ml/min or using an HPLC system (model LC-20AD; Shimadzu) equipped with a TSKgel Polyaminepak (Tosoh, Tokyo, Japan) with gradient elution at 40 °C at a flow rate of 0.4 ml/min. The running program for the HPLC system using the Shim-pack Amino-Na column was described previously (9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In the analysis using TSKgel Polyaminepak, two buffers, buffer A (18.6 mm trisodium citrate dehydrate, 400 mm sodium chloride, 20.8 mm HCl, 4% methanol, 0.0016% octanoic acid, 0.0156% Brij-35) and buffer B (93 mm trisodium citrate dehydrate, 2 m sodium chloride, 104 mm HCl, 20% methanol, 0.008% octanoic acid, 0.078% Brij-35), were used. The column was originally equilibrated with buffer A. After the sample was injected, the concentration of buffer B was kept at 0% for 5 min. Then it was increased to 100% and maintained until 30 min. Then the column was regenerated by 0.2 n NaOH from 30 to 35 min. After the regeneration step, the column was equilibrated again by buffer A from 35 to 45 min. o-Phthalaldehyde was used as the detection reagent (21Benson J.R. Hare P.E. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 619-622Crossref PubMed Scopus (786) Google Scholar) as described previously (3Suzuki H. Izuka S. Minami H. Miyakawa N. Ishihara S. Kumagai H. Appl. Environ. Microbiol. 2003; 69: 6399-6404Crossref PubMed Scopus (52) Google Scholar), and fluorescence was detected with a fluorescence detector (model RF-10AXL; Shimadzu) at an absorbance of 470 nm with excitation at 340 nm. Standard compounds were purchased from Nacalai Tesque (Kyoto, Japan) and Sigma-Aldrich except γ-Glu-Put, which was synthesized as described previously (9Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In our HPLC system equipped with the Polyaminepak, γ-Glu-Put, putrescine, cadaverine, and spermidine were eluted at 10.5, 17.5, 22.3, and 28.1 min, respectively. In the preparation of whole-cell samples, 1mlof A600 = 1 culture was centrifuged, and the pellet was washed with 1 ml of M9-glucose minimal medium (16Miller, J. H. (1992) A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Bacteria, pp. 263–274 and 437, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar). The washed pellet was resuspended in 0.2 ml of 5% trichloroacetic acid (v/v; Nacalai Tesque) and boiled in a boiling water bath for 15 min to break the cell. The suspension was centrifuged, the supernatant was applied to HPLC after filtration using Millex-LH (Millipore, Billerica, MA), and the precipitated protein was dissolved in 1 ml of 0.1 n NaOH. The protein concentration of the solution was measured by the Lowry method (22Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and the polyamine concentration of the cell was calculated as nmol/mg of protein.Assays for PuuA ActivityHPLC Method—γ-Glu-Put synthetase activity was determined by measuring the decrease of glutamate. A reaction mixture containing 10 mm monosodium glutamate, 10 mm putrescine dihydrochloride, 7.5 mm ATP, 30 mm MgCl2, and 100 mm imidazole-HCl buffer (pH 8.0) was incubated at 37 °C. After stopping the reaction by adding trichloroacetic acid (final concentration, 10%), the decreased glutamate by PuuA was quantitated by HPLC.Coupled Enzymatic Method—Another simple procedure used to determine the Km value of PuuA was based on the method described previously (23Kingdon H.S. Hubbard J.S. Stadtman E.R. Biochemistry. 1968; 7: 2136-2142Crossref PubMed Scopus (96) Google Scholar). The reaction mixture containing 100 mm imidazole-HCl buffer (pH 9.0), 10 mm monosodium glutamate, 100 mm putrescine dihydrochloride, 7.5 mm ATP, 25 mm MgCl2, 10 mm KCl, 1 mm phosphoenolpyruvate, 0.14 mm NADH, 5 units/ml pyruvate kinase (Oriental Yeast, Tokyo, Japan), 12.6 units/ml lactate dehydrogenase (Oriental Yeast), and 1 μg/ml PuuA was incubated at 25 °C. The change in absorbance at 340 nm due to oxidation of NADH was followed using a UV-visible spectrometer (model UV-1600PC; Shimadzu).Purification of PuuAPuuA was purified from a cell-free extract prepared from a 200-ml culture of SO97 by 0–40% ammonium sulfate precipitation and column chromatography using a HiTrap Blue column (column volume, 5 ml; GE Healthcare). During purification, the protein was basically dissolved in buffer C (20 mm imidazole-HCl (pH 8.0) and 1 mm MnCl2). After ammonium sulfate fractionation, the enzyme was dissolved in buffer C and dialyzed against the same buffer. The dialyzed enzyme solution was applied to a HiTrap Blue column equilibrated previously with buffer C. PuuA was eluted with a linear gradient formed between buffer C and 20 mm ATP in buffer C. PuuA was eluted when the concentration of ATP was 8.5–11 mm. The purified PuuA appeared as a single band on the SDS-PAGE gel (Fig. 3). This purification is summarized in Table 2. One unit of PuuA was defined as the amount of enzyme required to expend 1 μmol of glutamate/min under the condition of the PuuA assay using the HPLC described under “Assays for PuuA Activity.” Protein concentration was measured by the Lowry method (22Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar).FIGURE 3SDS-PAGE of purification steps of PuuA. The numbers above the gel correspond to those of Table 2 showing the purification steps. “M” indicates the prestained protein marker Broad Range (New England Biolabs, Ipswich, MA). Five micrograms of protein were applied to each lane. The concentration of acrylamide in the separation gel was 12.5%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Purification of PuuA from SO58Total proteinTotal activitySpecific activityYieldmgunitsunits/mg%1. Cell-free extract10.333.53.241002. Ammonium sulfate (0-40%)4.6921.54.5764.13. HiTrap Blue1.4618.612.755.6 Open table in a new tab Real Time RT-PCR AnalysisTotal RNA was extracted and purified using an RNA Mini kit (Qiagen, Valencia, CA) following the manufacturer's instructions. One microgram of total RNA was treated with DNase I (final concentration, 0.1 units/μl; amplification grade; Invitrogen). After adding EDTA (final concentration, 2.27 mm) to inactivate DNase I, cDNA was synthesized by an iScript cDNA Synthesis kit (Bio-Rad) from 1 μg of starting total RNA primed with the random primers included in the kit according to the manufacturer's instructions. Tth RNase H (final concentration, 0.52 units/μl; Toyobo) was added to the reaction mixture to remove RNA. puuA-specific primers, puuA-RT1 and puuART2, were designed to amplify 95-nucleotide fragments using Primer3 software. The real time PCR mixture (brought to a final volume of 10 μl with deionized water) contained 5 μl of iQTM SYBR Green Supermix (Bio-Rad), 3 pmol of each of the two primers, and a 2-μl cDNA sample in a 96-well optical reaction plate mounted in a DNA Engine Opticon Continuous Fluorescence Detection System (Bio-Rad). The thermal cycling conditions were as follows: 95 °C for 15 min and 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. To ensure the absence of nonspecific PCR products, melting curve analysis and agarose gel electrophoresis were performed after each run. The number of transcripts in a sample was determined by comparing the number of cycles (C) required for the reaction to reach a common threshold (t) with a plot of Ct values against the standard pKHG8. The relative expression levels of puuA compared with controls were calculated using Opticon (Bio-Rad). The relative amount of transcripts between samples was further standardized by amplification of the gapA gene as an internal control usi
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