1 N1-AMINOPROPYLAGMATINE: A NEW POLYAMINE PRODUCED

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1 JBC Papers in Press. Published on June 27, 2005 as Manuscript M413332200 N1-AMINOPROPYLAGMATINE: A NEW POLYAMINE PRODUCED AS A KEY INTERMEDIATE IN POLYAMINE BIOSYNTHESIS OF AN EXTREME THERMOPHILE, Thermus thermophilus * Mio Ohnuma1, Yusuke Terui1, Masatada Tamakoshi1, Hidemichi Mitome2, Masaru Niitsu3, Keijiro Samejima3, Etsuko Kawashima2, and Tairo Oshima1 1 From the Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, the 2School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan, and the 3 Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 530-0248, Japan. Running title: Aminopropylagmatine and a new polyamine biosynthetic pathway Address correspondence to: Tairo Oshima; Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan. Phone: 81-426-76-7134; Fax: 81-426-76-7145; E-mail: [email protected] Key words: agmatine, agmatine ureohydrolase, N1-aminopropylagmatine, polyamine, spermidine synthase, Thermus thermophilus Downloaded from http://www.jbc.org/ by guest on September 11, 2016 In the extreme thermophile Thermus thermophilus, a disruption mutant of a Polyamines play important roles in cell gene homologous to speB (coding for proliferation and cell differentiation. agmatinase = agmatine ureohydrolase) Common polyamines such as putrescine, accumulated N1-aminopropylagmatine spermidine and spermine are distributed (N -amidino-1,8-diamino-4-azaoctane, N8- 8 ubiquitously in cells and tissues at relatively amidinospermidine), a new compound, high concentrations (1, 2). while all other polyamines produced by the Thermus thermophilus, of which the wild-type strain were absent from the cells. genome project was completed using two Double disruption of speB and speE strains, HB8 and HB27 (Structural-Biological (polyamine aminopropyltransferase) Whole Cell Project resulted in disappearance of N1- http://www.srg.harima.riken.go.jp/thermus/j_ aminopropylagmatine and accumulation index.htm, 3), produces a variety of of agmatine. These results suggest that (1) polyamines including unusually long N1-aminopropylagmatine is produced from polyamines and branched ones (4, Fig. 9C). agmatine by the action of an enzyme coded These long and branched polyamines have a by speE, (2) N1-aminopropylagmatine is a marked effect of protecting and stabilizing metabolic intermediate in the biosynthesis nucleic acids (5, 6) and of activating cell-free of unique polyamines found in the polypeptide synthesis at high temperature (7, thermophile, and (3) N1- 8, 9). aminopropylagmatine is a substrate of the In many organisms, bacteria, yeast, SpeB homolog. They further suggest a new animals and plants, the first step of biosynthetic pathway in T. thermophilus, polyamine biosynthesis is production of by which polyamines are formed from putrescine by decarboxylation of L-ornithine agmatine via N1-aminopropylagmatine. To (Fig. 9A). An additional or alternative confirm our speculation, we purified the pathway of putrescine biosynthesis that is expression product of the speB homolog often seen in plants and sometimes in and confirmed that the enzyme hydrolyzes bacteria is decarboxylation of L-arginine N1-aminopropylagmatine to spermidine followed by hydrolysis of agmatine. but does not act on agmatine. Agmatine ureohydrolase or agmatinase, 1 Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

2 coded by the speB gene, catalyses this second (94C for 0.5 min, 55C for 0.5 min and 72C reaction. The next step is production of for 1 min) with LA Taq in GC buffer (Takara spermidine and spermine by the addition of Bio) using pairs of oligonucleotide primers an aminopropyl group to putrescine and listed in Table 2. Two pairs of primers, spermidine, respectively. This reaction is speBKpnUp5-speBHinUp3 and catalyzed by spermidine or spermine speBEcoDw5-speBXbaDw3, were used to synthase (putrescine/spermidine construct pSBKm; and a further two pairs, aminopropyltransferase) coded by the speE speEup5kpn-speEup3hin and gene (1). speEdw5xba-speEdw3sac, were used to To investigate the polyamine construct pESE. Genomic DNA of T. biosynthetic pathway in T. thermophilus, we thermophilus HB8 was used as the PCR constructed a disruption strain of the speB template. PCR products were digested with gene homolog of T. thermophilus. Disruption restriction endonucleases listed in Table 2 of the speB gene homolog resulted in drastic and cloned into pBluescriptSK+ or pBHTK reduction of triamines, longer and branched (14) to construct pSBUKm, pSBD, pSEUKm polyamines without accumulation of and pSED. The 3 half of speB and the agmatine, and in accumulation of an downstream region of speE were inserted Downloaded from http://www.jbc.org/ by guest on September 11, 2016 unknown compound. Double disruption of into pSBUKm and pSEUKm to prepare speB and speE gene homologs resulted in pSBKm and pSEKm, respectively. The HTK disappearance of this compound and region of pSEKm was replaced with the pyrE accumulation of agmatine in the cells. The gene to construct pSEPE. new compound was identified as N1- To make speE overexpression plasmid, aminopropylagmatine (N8-amidino-1,8- PCR was carried out for 25 cycles of 98C 8 diamino-4-azaoctane, N -amidinospermidine) for 0.5 min and of 72C for 3 min with by comparison with the chemically PfuTurbo DNA polymerase (Stratagene) synthesized authentic compound. In vitro using primers of sE5'NdeI and sE3'Hind. To reactions revealed that SpeE is responsible make speB overexpression plasmid, PCR was for the production of N1- carried out for 25 cycles (94C for 0.5 min, aminopropylagmatine, and SpeB converts 55C for 0.5 min and 72C for 1 min) with N1-aminopropylagmatine to spermidine. LA Taq in GC buffer using primers of sB5'NdeI and sB3'Hind (Table 2). Each PCR EXPERIMENTAL PROCEDURES products were digested with NdeI and HindIII and were cloned between the NdeI Strains and culture conditions The HindIII site of expression plasmid pET21c+ strains of T. thermophilus and Escherichia to make pESE8 and pESB8, respectively. coli and plasmids used in this study are listed All cloned nucleotide sequences were in Table 1. The rich growth and minimum determined to verify the fidelity of the media for T. thermophilus were as described amplified product to the original sequence previously (10). Leucine, isoleucine and using BigDye Terminator Ready Reaction uracil (50 mg/ml each) were included in the Premix (Perkin-Elmer) and an ABI PRISM minimum medium. Media were solidified as 377 DNA Sequencing System (Perkin-Elmer described (11). Applied Biosystems). Construction of plasmids All Gene disruption To obtain speB and nucleotide sequences of T. thermophilus HB8 speE gene homolog disruption strains of T. used in this study were kindly provided by Dr. thermophilus, the thermophile host was Seiki Kuramitsu of Osaka University. genetically transformed as described Construction of pSBKm and pSEPE is shown previously (11, 16). All genetic in Fig. 1. PCR was carried out for 25 cycles recombinations were confirmed by Southern 2

3 blot analysis. buffer (100 mM HEPES-NaOH (pH 7.5), 5 HPLC Cells of T. thermophilus were mM MgCl2, 5 mM dithiothreitol, 40 mM collected from 250 ml of overnight culture pyridoxal phosphate and 1mM agmatine) (about A590 = 0.3-0.4) in minimum medium at were disrupted by sonication. After removing 70C. Three-fold volume of 10% TCA was the cell debris by centrifugation (30 krpm, 20 added to the collected cell paste, and the min), the cell extract was heated for 15 min mixture was vortexed thoroughly to extract at 75C and the denatured protein was cellular polyamines. Appropriately diluted removed by centrifugation (30 krpm, 20 min). samples were analyzed to determine Heat treatment was repeated twice. The polyamine composition using a CK-10S supernatant was fractionated by 30-50% column (8.0 x 70 mm, Mitsubishi Kasei) as saturated ammonium sulfate. The precipitate described previously (17). All samples were collected by centrifugation was suspended in subjected to GC1 and GC-MS analyses to SB buffer. The enzyme was further purified confirm polyamine species, as described by by a Hiprep QXL (Amasham Biosciences) Niitsu et al. (18). column chromatography. Partially purified Overexpression and purification of SpeB was stored at 4C as a suspension in speE and speB gene products E. coli BL21- 60% saturated ammonium sulfate in 50 mM Downloaded from http://www.jbc.org/ by guest on September 11, 2016 CodonPlus(DE3)-RP carrying pESE8 or sodium phosphate buffer (pH 7.0) until use. pESB8 were grown in 10 l of 2xYT medium Protein determination Protein at 37C. When apparent absorbance at 600 concentrations were estimated with BCA nm reached to 0.3, isopropyl-b-D- Protein Assay Reagent Kit (PIERCE) using thiogalactopyranoside was added to the bovine serum albumin as a standard. culture to a final concentration of 0.5 mM Enzymatic reactions To determine Km and incubation was continued for further two and kcat of SpeE, the aminopropyltransferase hours. For purification of SpeE, cells were assay was performed as described in collected by centrifugation and suspended in literature (19), except pH was 9.0 and 10 ml of 50 mM Tris-HCl (pH 7.5), 1 mM agmatine was used as substrate instead of EDTA followed by cell disruption with a putrescine. The [methyl-14C] decarboxylated sonic oscillator. After removing the cell S-adenosylmethionine (dcSAM) was debris by centrifugation (30 krpm, 20 min), synthesized by a method of Samejima and the cell extract was heated for 15 min at 75C colleagues (Samejima, unpublished). and the denatured protein was removed by To confirm the reaction product of centrifugation (30 krpm, 20 min). Heat SpeE, a reaction mixture consisting of 100 treatment was repeated twice. The proteins mM Tris-HCl (pH 9.0 at 37C), 5 mM precipitated by 50% saturated ammonium dithiothreitol, 2 mM agmatine, 337.5 mM sulfate were collected by centrifugation, and dcSAM and 32 mg of purified SpeE in a final suspended in 20 ml of 50 mM Tris-HCl (pH volume of 2 ml was incubated at 37C 7.5), 1 mM EDTA. Then the enzyme was overnight. Polyamines were isolated using a further purified by a Hiprep QXL column Dowex 50W x 4 (Muromachi Technos Co. (Amasham Biosciences) and by a Resource Ltd) column chromatography. Fractions HIC PHE column (Amasham Biosciences) containing polyamines which react with O- chromatographies. SpeE thus obtained was phtalaldehyde were collected and homogeneous on a SDS-gel electrogram and concentrated. was stored at 4C as a suspension in 60% For identification of chemical reaction saturated ammonium sulfate in 50 mM catalyzed by SpeB, a reaction mixture sodium phosphate buffer (pH 7.0) until use. consisting of 100 mM HEPES-NaOH (pH For purification of SpeB, cells were 7.4), 5 mM MgCl2, 5 mM dithiothreitol, 40 corrected and suspended in 10 ml of SB mM pyridoxal phosphate, 50 mg of partially 3

4 purified SpeB, and 40 mg of dried reaction Bannard et al (21), except that a lager amount product of SpeE described above or 50 mg of of hydrobromic acid was added to the chemically synthesized N1- reaction mixture prior to crystallization by aminopropylagmatine in a final volume of 50 adding ether. The yield was 68%. 1H-NMR ml, was incubated at 65C for 1 h. In another (CDCl3): 9.25 (2H, bs), 7.77 (2H, bs), 6.15 experiment, 40 mg of agmatine was added (1H, s), 2.82 (3H, s), 2.26 (3H, s). instead of N1-aminopropylagmatine to 3-Aminopropylagmatine investigate whether or not SpeB possesses pentahydrochloride (6) A stirred solution of agmatinase activity. The reaction product was 1-phtalimidopropyl-1,4-butandiamine analyzed by HPLC. dihydrobromide (4) (0.09 g, 0.21 mmol) and Chemical Synthesis 1-guanyl-3,5-dimethyl pyrazole Mono(benzyloxycarbonyl)-1,4- hydrobromide (5) (0.045 g, 0.21 mmol) in 2 butandiamine hydrochloride (3) This ml of dry ethanol was refluxed for 11 h in the compound was prepared as a chloride by the presence of NaOH (0.025 g, 0.63 mmol). method of Lawson et al. (20), except for the After removal of NaBr, the reaction product use of 1,4-butanediamine instead of 1,3- was washed with ether and dried. The residue propandiamine to prepare was refluxed for 24 h with 5 ml of Downloaded from http://www.jbc.org/ by guest on September 11, 2016 bis(benzyloxycarbonyl)-1,4-butandiamine (2). concentrated hydrochloric acid. The reaction The yield was 32%, m.p. 189-192C. Lawson mixture was applied to a small column of et al. (20) report m.p. 191-193C. 1H-NMR Dowex 50W-X4 (H+ form) and eluted with 6 (CDCl3): 7.26-7.35 (5H, m), 5.08 (3H, s), M hydrochloric acid. Fractions of 30 ml were 3.16-3.23 (2H, m), 2.68-2.72 (2H, m), 1.44- collected and examined by analytical HPLC, 1.56 (6H, m). 13C-NMR (CDCl3): 156.1, and those containing the desired compound 136.4, 128.2, 127.8, 77.4, 77.0, 76.6, 66.5, were combined and evaporated to dryness 41.8, 40.9, 30.9, 27.5. under reduced pressure at about 40C. A pale 1-Phthalimidopropyl-1,4-butandiamine yellow oil was obtained (0.06 g, 77%). 1H- dihydrobromide (4) A stirred solution of NMR (400 MHz, D2O) d: 1.69 (2H, m), 1.80 mono(benzyloxycarbonyl)-1,4-butandiamine (2H, m), 2.13 (2H, m), 3.14 (6H, m), 3.25 hydrochloride (3) (1.00 g, 3.87 mmol) and N- (2H, t). 13C NMR (75 MHz, D2O) d: 22.88, (3-bromopropyl)phthalimide (1.04 g, 3.87 23.78, 25.10, 36.60, 40.49, 44.52, 47.32, mmol) in 13 ml of acetonitrile was refluxed 156.77. HRMS: (TOF MS ES+) Calcd. for at 40C for 19 h in the presence of KF-Celite C8H22N5 (M + H)188.1875, Found 188.1886; (0.6 g). The product was filtered, washed Calcd. for C8H23N5 (M + 2H)189.1953, with ether, and dried. The residue was treated Found 189.1953; Calcd. for C8H24N5 (M + with 10 ml of hydrobromic acid (30-32%) in 3H)190.2032, Found 190.2007. acetic acid for 1 h at room temperature, then added to dry ether. The 1-phthalimidopropyl- RESULTS 1,4-butandiamine dihydrobromide precipitated was washed with dry ether and To elucidate the biosynthetic pathway evaporated to dryness to yield a white solid of T. thermophilus, we searched for gene (0.90 g, 53%). This solid was hydroscopic homologs of known polyamine biosynthetic and was recrystalized from methanol. 1H- enzymes in the T. thermophilus genome. NMR (CD3OH): 7.80-7.89 (4H, m), 3.79- Homologs of arginine decarboxylase (speA, 3.84 (2H, t), 2.98-3.14 (6H, m), 2.07-2.13 ORF number of T. thermophilus HB8 and (2H, m), 1.78-1.80 (4H, m). HB27 (3) is TT0340 and TTC1277, 1-Guanyl-3,5-dimethyl pyrazole respectively), agmatine ureohydrolase (speB, hydrobromide (5) This compound was HB8, TT0338; HB27, TTC0764), two S- prepared as a nitrate by the method of adenosylmethionine decarboxylases (speD, 4

5 HB8, TT1493 and 1749 ;HB27, TTC0473 weight was estimated as 775 based on mass and TTC1093) and spermidine synthase numbers of molecular fragments observed on (speE, HB8, TT0339; HB27, TTC0472) were GC-MS spectrum shown in Fig. 5B. This found, but a homolog of the ornithine corresponds to the molecular weight of N1- decarboxylase gene (speC) was not. aminopropylagmatine bound to three Alignment of the amino acid sequences heptafluorobutyryl molecules. Since the gene of speB gene products is shown in Fig. 2. The coding for aminopropyltransferase is present amino acid sequence deduced from the T. in MOSB, the major unknown polyamine of thermophilus speB gene homolog showed 29- MOSB can be predicted to be N1- 32% similarity to those of other organisms. aminopropylagmatine. Two additional The histidine residue that is critical for unknown polyamines (X, Y in Fig. 4B) could catalytic activity in the E. coli enzyme (22) not be identified because their amounts were and the manganese ion-binding residues (23) too small. are conserved in all amino acid sequences To confirm the identity of polyamine A, examined. authentic N1-aminopropylagmatine was To ascertain whether polyamine chemically synthesized (Fig. 6). In HPLC synthesis starts from ornithine or arginine in analyses, the retention time of the Downloaded from http://www.jbc.org/ by guest on September 11, 2016 the thermophile, we first disrupted the speB synthesized N1-aminopropylagmatine was gene homolog of T. thermophilus. The identical to that of polyamine A (data not disruption strain, named MOSB, was shown). constructed by insertion of the HTK gene Having identified polyamine A as N1- into the speB gene homolog of TTY1. When aminopropylagmatine, we next disrupted the MOSB was cultivated in minimum medium gene of polyamine aminopropyltransferase at 70C, the disruption showed no effect on (speE homolog) of MOSB. To construct the growth (doubling time of MOSB was 7.2 h speB and speE double-disruption strain, we while that of TTY1 was 4.9 h, Fig. 3). disrupted the speB homolog by inserting the Growth on a minimum medium plate at 75C HTK gene of T. thermophilus (DspeE) whose also showed little difference between MOSB speE homolog had been replaced with the and TTY1 (data not shown). However, pyrE gene. The double disruptant was named disruption of the speB gene homolog resulted T. thermophilus MOSBE. MOSBE showed in significant defect of growth at 78C in defective growth at 70C in minimum minimum medium (Table 3). In addition, the medium (doubling time was 12.8 h, Fig. 3) MOSB colony was pale yellow, while the and significantly defective growth at 78C TTY1 colony exhibited a bright yellow color (Table 3). Like that of MOSB, the MOSBE (data not shown). colony was pale yellow (data not shown). As Intracellular polyamine composition of shown in Fig. 4, agmatine was accumulated MOSB grown in minimum medium at 70C in MOSBE cells, and N1- was analyzed by HPLC. Most polyamines aminopropylagmatine, the major polyamine found in the cells of TTY1 (Fig. 4A) fell of MOSB, was drastically diminished. A below the limit of detection by our method in small amount of cadaverine was detected, but MOSB (Fig. 4B). At the same time, an other polyamines, especially long and unknown polyamine designated as polyamine branched polyamines, were undetectable. A in Fig. 4 was accumulated. Cadaverine, These results indicated that the major agmatine and two unknown polyamines (X, polyamine biosynthesis in T. thermophilus Y) were concomitantly detected as minor starts from arginine, not from ornithine. A compounds. Fig. 5A shows the results of GC new polyamine, N1-aminopropylagmatine, analysis of polyamines in MOSB. The major produced from agmatine by the speE gene peak occurred at 9.3 min, and its molecular homolog, plays a key role as a metabolic 5

6 intermediate in polyamine biosynthesis in the least a year. The kinetic parameters of SpeE thermophile. We therefore concluded that the were determined by Hanes-Woolf plot. Since T. thermophilus speB gene homolog codes for dcSAM decompose at high temperature in N1-aminopropylagmatine ureohydrolase, and alkaline condition (24), reactions for the speE gene homolog codes for agmatine obtaining kinetic parameters were performed aminopropyltransferase. at 37C, pH 9 with various concentrations of Although MOSB could not grow in agmatine (0.5 - 2 mM) and 38 mM of dcSAM. minimum medium at 78C (Table 3), the The enzymatic reaction was performed in one growth recovered when 250 mM spermidine tube and 100 ml of reaction mixture was was added to the medium. The polyamine sampled to stop reaction at 0.5, 1, 5, 10, 15 composition of MOSB grown at 78C is and 20 min. The Km value for agmatine was shown in Fig. 7. A long polyamine, 0.77 mM. The kcat was 0.37 sec-1 when homocaldopentamine, was accumulated as agmatine was used as substrate. the major component in these cells. A small The partially purified SpeB migrated as amount of quaternary branched polyamine, a single band (32.5 kDa) on a SDS-PAGE as tetrakis(3-aminopropyl)ammonium, was also shown in Fig. 8B. present. N1-aminopropylagmatine was not As shown in Fig. 8Ca, agmatine was Downloaded from http://www.jbc.org/ by guest on September 11, 2016 produced under these conditions, suggesting converted to N1-aminopropylagmatine by that the accumulation of long polyamines purified SpeE. In addition, purified SpeB represses its production. The finding that converted the reaction product of SpeE into MOSB produced long and branched spermidine in vitro (Fig. 8Cb). Chemically polyamines in medium supplemented with synthesized N1-aminopropylagmatine was spermidine suggests that conversion of N1- also converted to spermidine by SpeB (see aminopropylagmatine to spermidine is an Fig. 8Ce and f). SpeB could not utilize essential step in the synthesis of long and agmatine as a substrate (Fig. 8Cc, d). These branched polyamines, and that the speB gene results confirmed that SpeE converts product of T. thermophilus is responsible for agmatine to N1-aminopropylagmatine, and the reaction. SpeB converts N1-aminopropylagmatine to To confirm that SpeE converts spermidine. agmatine to N1-aminopropylagmatine and that SpeB uses N1-aminopropylagmatine as a DISCUSSION substrate to produce spermidine, we purified SpeE and partially purified SpeB to perform T. thermophilus produces no fewer in vitro enzymatic reactions. A typical than sixteen polyamines (Fig. 9C), and its purification procedure of SpeE was long and branched polyamines have been summarized in Table 4. SpeE was recovered suggested to play important roles in from the soluble fraction and purified by an thermophily (4, 8, 17). However, the starting anion exchange chromatography and a point of polyamine biosynthesis of this hydrophobic interaction chromatography. bacterium remains to be elucidated. We could Purity of SpeE by SDS-PAGE was shown in not find a gene homolog coding for ornithine Fig. 8A. The purified SpeE migrated as a decarboxylase (speC gene) in the T. single band (36 kDa). The final preparation thermophilus genome, even though Pantazaki had a specific activity of 2.4 mmol/min/mg et al. have reported purification and proteins, 51-fold of that of the crude enzyme. characterization of ornithine decarboxylase The purified enzyme was significantly of T. thermophilus (25). Some bacteria such stable: little loss of activity observed after as Rhodopirellula baltica and Selenomonas storage at 4C in 50 mM sodium phosphate ruminantium contain a homolog of buffer (pH 7.0), 1 M ammonium sulfate for at eukaryotic ornithine decarboxylase with dual 6

7 specificity on lysine and ornithine (26, 27). and is suppressed under normal conditions, We could not find a gene homolog coding for but is expressed when cellular polyamines eukaryotic ornithine decarboxylase. We disappear, as has been reported for E. coli speculate two possible explanations for the lysine decarboxylase. In E. coli, expression discrepancy between the report for ornithine of the lysine decarboxylase gene is decarboxylase and genome sequence data of suppressed by putrescine and spermidine (28). T. thermophilus: either the gene coding for Similarly cadaverine may not be present in ornithine decarboxylase has no homology to TTY1 (Fig. 4). It is worthy of note that only speC genes from other organisms, or an agmatine and cadaverine support the growth amino acid decarboxylase, such as arginine of the thermophile at lower temperature. decarboxylase or lysine decarboxylase, has Other polyamines, however, are essential for broad substrate specificity and accepts growth at higher temperatures. As shown in ornithine as a substrate. Fig. 7, when spermidine was added to the Phenotypes of disruption strains of minimum medium, MOSB cells produced polyamine biosynthetic genes In the present long and branched polyamines and were able study, we constructed an speB gene homolog- to survive at 78C. Therefore, long and disruption mutant of T. thermophilus branched polyamines are essential for growth Downloaded from http://www.jbc.org/ by guest on September 11, 2016 (MOSB) in order to clarify the first step of at over 75C. In the wild-type strain, cellular polyamine biosynthesis. MOSB exhibited content of these polyamines increased with significantly defective growth at 78C in the rise in the growth temperature (4). minimum medium but normal growth at 70C Both MOSB and MOSBE formed pale (Table 3 and Fig. 3). Polyamines found in yellow colonies, while the parent strain, MOSB cells (Fig. 4) were cadaverine, TTY1, forms a bright yellow colony. This agmatine and N1-aminopropylagmatine, suggests that one or more long and/or compared with of 16 or more found in TTY1. branched polyamines are required for (1) In addition, two larger unknown polyamines gene transcription, or (2) translation, or (3) were found. Judging from their peak enzymatic activity of the enzyme(s) involved positions in HPLC, these compounds may be in the dye synthesis. The intracellular content aminopropylated derivatives of N1- of the yellow pigments, carotenoids, which aminopropylagmatine. Their identifications are known act as antioxidants increase when will be the subject of future studies. These the cells are grown at higher temperature (29). observations indicate that T. thermophilus We speculate that MOSB and MOSBE are can grow at up to 75C with unable to grow at over 75C due to the aminopropylagmatine and other suppression of carotenoid synthesis and the aminopropylated derivatives that could lack of antioxidant. substitute for long and branched polyamines N1-Aminopropylagmatine In the in biochemical reactions at high temperature. present study, we identified the major When speB and speE gene homologs unknown polyamine accumulated in MOSB of T. thermophilus were disrupted, MOSBE as N1-aminopropylagmatine. A related had defective growth even at 70C (Fig. 3). polyamine in leech, hirudonine (G3-4N), was Agmatine accumulated in MOSBE cells and reported by Robin et al. (30). Chemically the levels of N1-aminopropylagmatine and synthesized N1-aminopropylagmatine also other polyamines diminished (Fig. 4). A appears in the literature: it was named N8- small amount of cadaverine was also detected guanylspermidine and is known to be a for the first time in the cells of T. strong inhibitor of deoxyhypusine synthase thermophilus. These observations suggest (31). However, this is the first time that N1- that a gene coding for lysine decarboxylase is aminopropylagmatine has been found as a present in the T. thermophilus chromosome natural compound. 7

8 Enzymatic activity of SpeE When present results suggest the danger of utilizing SpeE was incubated with agmatine and only sequence homology for genome dcSAM, only a small amount of annotation. aminopropylagmatine was formed as shown Based on reverse genetic analyses, we in Fig. 8Ca. This is not due to low catalytic propose a new polyamine biosynthetic activity of SpeE protein, but probably due to pathway in T. thermophilus. As shown in Fig. instability of dcSAM under the conditions 9B, biosynthesis of major polyamines in T. employed. The kcat value (0.37 sec-1) of the thermophilus starts only from arginine, which thermophile SpeE is by no means inferior to is decarboxylated to form agmatine. An those of other organisms even at 37C (19, aminopropyl group is added to agmatine by 32). Km for agmatine (0.77 mM at 37C) of SpeE to form N1-aminopropylagmatine. the present thermophile Finally N1-aminopropylagmatine is aminopropyltransferase is smaller than the hydrolyzed to spermidine by SpeB. In this reported Km for putrescine (20 mM at 37C) new polyamine biosynthetic pathway, of Thermotoga maritima spermidine synthase spermidine is synthesized without the (32). On the other hand, dcSAM is known to production of putrescine. It would be possible be unstable at alkaline pH (24). To reduce the to find this polyamine pathway in other Downloaded from http://www.jbc.org/ by guest on September 11, 2016 rate of spontaneous degradation of dcSAM, organisms. the experiment shown in Fig. 8Ca was As shown in Fig. 4, MOSB, which carried out at 37C. The reaction mixture was could not produce spermidine from N1- incubated overnight but the reaction might aminopropylagmatine, failed to produce long terminate earlier due to shortage of dcSAM. and branched polyamines. Such polyamines In the thermophile cells, some mechanisms, were produced in MOSB cells only after such as coupling of reactions, may exist to addition of spermidine to the minimum prevent the degradation of dcSAM at high medium (Fig.7). Therefore, the conversion of temperature. N1-aminopropylagmatine to spermidine is Biosynthetic pathway of polyamines in essential for production of long and branched T. thermophilus Based on the intracellular polyamines in T. thermophilus. polyamine composition of MOSB and Agmatine and aminopropylagmatine MOSBE (Fig. 4), we conclude that (1) major were not detected in the wild-type strain (Fig. polyamines are mainly derived from arginine 4A). The formation of spermidine in wild- rather than ornithine, (2) SpeE produces N1- type cells may be so rapid that only trace aminopropylagmatine from agmatine, and (3) amounts of these intermediates exist. In SpeB converts N1-aminopropylagmatine to MOSB cells cultivated in the presence of spermidine. spermidine, thermospermine should be We confirmed that SpeB acts on N1- present as a precursor of homocaldpentamine, aminopropylagmatine but not on agmatine but it was not detected by HPLC analysis (Fig. 8Ca-f). Sequence homology between T. (Fig. 7). Thermospermine has been identified thermophilus speB and speB of other in the cells of T. thermophilus, as well as organisms is not high as shown in Fig. 2, and homocaldopentamine and other long this low similarity would be reflected in the polyamines. In MOSB, it seems that the different substrate specificity of the aminopropylation of thermospermine is fast thermophile enzyme. It would be interesting enough that only a trace amount of to compare the tertiary structures of SpeB of thermospermine is present. These findings T. thermophilus and speB gene products of suggest that a delicate regulatory network other organisms that differ in substrate operates for polyamine biosynthesis of T. specificity. We are currently attempting to thermophilus. In the presence of spermidine, crystallize SpeB for structural analyses. The production of aminopropylagmatine was 8

9 suppressed in MOSB (Fig. 7). This finding HB27, TTC1205) is present in the T. also suggests the existence of a regulatory thermophilus genome (both strains HB8 and system in T. thermophilus. HB27); and this gene might be involved in In the new pathway we have proposed, sym-homospermidine synthesis. However, no polyamine synthesis starts with even trace of sym-homospermidine was decarboxylation of arginine. Spermidine is detected in MOSB, which accumulated N1- produced directly from N1- aminopropylagmatine in the cells (Fig. 4, 5). aminopropylagmatine, but not from This is consistent with the fact that N1- putrescine. The pathway is unique in that aminopropylagmatine acts as a putrescine is not involved in the biosynthesis deoxyhypusine inhibitor (31). To clarify the of other polyamines. However, small details of polyamine metabolism in T. amounts of putrescine, sym-homospermidine, thermophilus, further reverse genetic and other polyamines containing aminobutyl analyses as well as enzymatic investigations groups are found in wild-type cells, are necessary. In addition, detailed properties suggesting that genes other than speB are also and structures of SpeB, D, and E are being involved in polyamine metabolism of T. studied in our laboratory. Structural data for thermophilus. In this context, it is noteworthy SpeE has been deposited in a database (PDB Downloaded from http://www.jbc.org/ by guest on September 11, 2016 that a homolog of a gene coding for code 1UIR). deoxyhypusine synthase (33, HB8, TT0337; REFERENCES 1. Tabor, C. W., and Tabor, H. (1984) Ann. Rev. Biochem. 53, 749-790 2. Igarashi, K., and Kashiwagi, K. (2000) Biochem. Biophys. Res. Commun., 271, 559-564 3. Henne, A., Bruggemann, H., Raasch, C., Wiezer, A., Hartsch, T., Liesegang, H., Johann, A., Lienard, T., Gohl, O., Martinez-Arias R., Jacobi, C., Starkuviene, V., Schlenczeck, S., Dencker, S., Huber, R., Klenk, H. P., Kramer, W., Merkl, R., Gottschalk, G., and Fritz, H. (2004) Nat. Biotechnol. 22, 547-53 4. Oshima, T. (1989) in The Physiology of Polyamines CRC press Inc Volume II 36-46 5. Kirino, H., Kuwahara, R., Hamasaki, N., and Oshima, T. (1990) J. Biochem. 107, 661-665 6. Terui, Y., Ohnuma, M., Hiraga, K., Kawashima, E., Oshima, T. (2005) Biochem J. 388, 427-433 7. Ohno-Iwashita, Y., Oshima, T., and Imahori, K. (1975) Arch. Biochem. Biophys. 171, 490- 499 8. Uzawa, T., Hamasaki, N., and Oshima, T. (1993) J. Biochem., 114, 478-486 9. Kakegawa, T., Takamiya, K., Ogawa, T., Hayashi, Y., Hirose, S., Niitsu, M., Samejima, K., and Igarashi, K. (1988) Arch. Biochem. Biophys. 261, 2, 250-256 10. Tanaka, T., Kawano, N., and Oshima, T. (1981) J. Biochem. (Tokyo) 89, 677-682 11. Takada, T, Akanuma, S, Kotsuka, T, Tamakoshi, M, Yamagishi, A., and Oshima, T. (1993) Appl. Environ. Microbiol. 59, 2737-2739 12. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 103-119 13. Tamakoshi, M., Yaoi, T., Oshima, T., and Yamagishi, A. (1999) FEMS Microbiol. Lett. 173, 431-437 14. Nureki, O., Shirouzu, M., Hashimoto, K., Ishitani, R., Terada, T., Tamakoshi, M., Oshima, T., Chijimatsu, M., Takio, K., Vassylyev, D. G., Shibata, T., Inoue, Y., Kuramitsu S., and Yokoyama S. (2002) Acta Cryst. 58, 1129-1137 15. Tamakoshi, M., Uchida, M., Tanabe, K., Fukuyama, S., Yamagishi, A., and Oshima, T. (1997) J. Bacteriol. 179, 4811-4814 9

10 16. Koyama, Y., Hoshino, T., Tomizawa, N., and Furukawa, K. (1986) J. Bacteriol. 166, 338- 340 17. Oshima, T. (1983) in Method in Enzymology, Academic Press 94, 401-411 18. Niitsu, M., Samejima, K., Matsuzaki, S., and Hamana, K. (1993) J. Chromatogr. 641, 115- 123 19. Raina, A., Eloranta, T., and Pajula, R. (1983) in Method in Enzymology, Academic Press 94, 257-260 20. Lawson, W. B., Leafer, M. D., Tewes, A., and Rao, G. J. S. (1968) Hoppe-Seylers Z. Physiol. Chem., 349, 251-262 21. Bannard, R. A. B., Casselman, A. A., Cockburn, W. F., and Brown, G. M. (1958) Canad. J. Chem. 36, 1541-1549 22. Carvajal, N., Olate, J., Salas, M., Lpez, V., Cerpa, J., Herrera, P., and Uribe, E. (1999) Biochem. Biophys. Res. Comm. 264, 196-200 23. Perozich, J., Hempel, J., and Morris, S. M. Jr. (1998) Biochim. Biophys. Acta. 1382, 23-37 24. Zappia, V., Galletti, P., Oliva, A., and de Santis, (1977) Anal Biochem. 79, 535-43 25. Pantazaki, A. A., Anagnostopoulos, C. G., Lioliou, E. E., and Kyriakidis, D. A. (1999) Mol. Cell Biochem. 195, 55-64 Downloaded from http://www.jbc.org/ by guest on September 11, 2016 26. Glkner, F. O., Kube, M., Bauer, M., Teeling, H., Lombardot, T., Ludwig, W., Gade D., Beck, A., Borzym, K., Heitmann, K., Rabus, R., Schlesner, H., Amann, R., and Reinhardt, R. (2003) PNAS 100, 8298-8303 27. Takatsuka, Y., Yamaguchi, Y., Ono, M., and Kamio, Y. (2000) J. Bacteriol. 182, 6732-6741 28. Wertheimer S. J., and Leifer Z. (1983) Biochem. Biophys. Res. Commun. 114, 882-888 29. Ray, P. H., White, D. C., and Brock, T. D. (1971) J. Bacteriol 108, 227-235 30. Robin, Y., Audit, and C., Landon, M. (1967) Comp. Biochem. Physiol. 22, 787-797 31. Jakus, J., Wolff, E. C., Park, M. H., and Folk, J. E. (1993) J. Biol. Chem. 268, 13151-13159 32. Korolev, S., Ikeguchi, Y., Skarina, T., Beasley, S., Arrowsmith, C., Edwards, A., Joachimiak, A., Pegg, A., E., and Savchenko, A. (2002) Nat struct biol. 9, 27-31 33. Park, M. H., Wolff, E. C., and Folk, J. E. (1993) Biofactors 4, 95-104 FOOTNOTES * We are grateful to Dr. S. Kuramitsu (Osaka University) for providing nucleotide sequences of T. thermophilus HB8. We thank Mses. Mizue Nagai, Hiromi Ito, Marie Kajino and Aya Chitose for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research (No. 11794038 and 16657040) and by Grants-in-Aid for Promoting Bioventures in Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. 1 The abbreviations used are: HTK, highly thermostable kanamycin nucleotidyltransferase; GC, gas chromatography; GC-MS, gas chromatography mass spectrometry; dcSAM, decarboxylated S-adenosylmethionine; HEPES, N-2- hydroxyethylpiperazine-N-2-ethanesulfonic acid. FIGURE LEGENDS Fig. 1 Construction of plasmids used for gene disruption of speB and speE. See Experimental and Procedures for details. (A) Construction of pSBKm. (B) Construction of pSEPE. 10

11 Fig. 2 Multisequence alignment of speB gene homologs. Amino acid sequences of speB gene homologs were aligned using the Clustal W program with a gap opening penalty of 10 and gap extension penalty of 0.05. Amino acid sequences were obtained from GenBank database searches performed with the BLAST program through the homepages of the National Center of Biotechnology Information. Identical and similar residues are shaded with dark and light gray, respectively. The arrow points to the histidine residue of E.coli SpeB critical for catalytic activity, and arrowheads point to the residues involved in the binding of manganese ion. The abbreviations denote the following bacterial species: Tth_HB8, Thermus thermophilus HB8, Tth_HB27, Thermus thermophilus HB27; Eco, Escherichia coli; Bsu, Bacillus subtilis; and Sto, Sulfolobus tokodaii. Fig. 3 Effects of intrinsic polyamines on growth of T. thermophilus in minimum medium at 70C. Filled diamonds, TTY1; open circles, MOSB (speB::HTK strain); and filled triangles, MOSBE (speB::HTK, DspeE strain). Fig. 4 Polyamine composition of T. thermophilus. Polyamines extracted from cells of (A) TTY1 (harvested when A590 = 0.4), (B) MOSB (speB::HTK strain, harvested when A590 = 0.31) Downloaded from http://www.jbc.org/ by guest on September 11, 2016 and (C) MOSBE (speB::HTK, DspeE strain, A590 = 0.33) were analyzed by HPLC. Dilutions of each sample are indicated in the figure. A, X and Y are unknown polyamines. Unknown polyamine A was determined to be N1-aminopropylagmatine. Put, putrescine; Cad, cadaverine; Spd, spermidine; Agm, agmatine; Thm, thermine; Spm, spermine and thermospermine (these two polyamines can not be separated on the HPLC employed in the present study); Cdp, caldopentamine; Hcdp, homocaldopentamine; Taa, tetrakis(3-aminopropyl)ammonium. Fig. 5 GC and GC-MS analyses of polyamines in MOSB. (A) GC chromatogram of a MOSB cell extract after heptafluorobutyrization and concentration. (B) GC-MS analysis of the peak eluted at 9.32 min. Polyamines identified by GC are indicated in abbreviated forms expressing the numbers of methylene chain units. Parentheses represent a branched polyamine. 33, norspermidine; 34, spermidine; 3(3)4, N4-aminopropylspermidine. Fig. 6 Scheme for chemical synthesis of N1-aminopropylagmatine. Fig. 7 Polyamine composition of MOSB strain grown in minimum medium supplemented with 250 mM spermidine at 78C. Polyamines extracted from cells of MOSB (speB::HTK strain, harvested when A590 = 0.3) were diluted ten-fold and analyzed by HPLC. Inset shows HPLC elution profile of the same sample without dilution. Spd, spermidine; Hspd, sym- homospermidine; Agm, agmatine; Thm, thermine; Spm, spermine and thermospermine (see legend for Fig. 4); Hcdp, homocaldopentamine; Taa, tetrakis(3-aminopropyl)ammonium. Fig. 8 In vitro polyamine syntheses using purified SpeE and SpeB. (A) Purification of recombinant SpeE. Samples were separated by SDS-PAGE on 12% gel. M, molecular marker (SDS-PAGE standard low, Bio-Rad); lane 1, total cell lysate; lane 2, 1st heat treatment; lane 3, 2nd heat treatment; lane 4, ammonium sulfate precipitation; lane 5, Hiprep QXL fraction; lane 6, Resource HIC PHE fraction. (B) Purification of recombinant SpeB. Samples were separated by SDS-PAGE on 9.4% gel. M, molecular marker; lane 1, supernatant after sonication; lane 2, supernatant of heat treatment; lane 3, 40% ammonium sulfate precipitation; lane 4, Hiprep QXL fraction; (C) In vitro reactions of purified SpeE and SpeB. a: Polyamine composition after agmatine was incubated with SpeE and dcSAM. For catalytic activities of SpeB, substrates used 11

12 are the products of SpeE enzymatic reaction (a, b), agmatine (c, d) or chemically synthesized N1-aminopropylagmatine (e, f). After 0 min and 60 min, reaction mixtures were analyzed by HPLC. Spd, spermidine; Agm, agmatine; Aminopropyl-agm, N1-aminopropylagmatine. Fig. 9 Polyamine biosynthetic pathway. (A) Reported polyamine biosynthetic pathway for bacteria and plants. (B) Proposed polyamine biosynthetic pathway of T. thermophilus. (C) Structures of Polyamines. * represents polyamines that were identified in wild type T. thermophilus cells. Downloaded from http://www.jbc.org/ by guest on September 11, 2016 12

13 Table 1 Bacterial strains and plasmids used in this study Strain or DNA Description / genotype Source or reference Strain E. coli JM109 Takara Bio, (12) BL21-CodonPlus(DE3)-RP Novagen T. thermophilus TTY1 T. thermophilus HB27 but DleuB DpyrE (13) MOSB T. thermophilus TTY1 but speB::HTK This study MOSE T. thermophilus TTY1 but DspeE This study MOSBE T. thermophilus MOSE but speB::HTK This study Plasmids pBlueScriptSK+ Toyobo pBHTK Plasmid carrying HTK gene (14) pINV Plasmid carrying pyrE gene (15) pSBUKm Ampr, HTK This study Downloaded from http://www.jbc.org/ by guest on September 11, 2016 pSBD Ampr This study pSBKm Ampr, speB::HTK This study pSEUKm Ampr, HTK This study pSED Ampr This study pSEKm Ampr, DspeE, HTK This study pSEPE Ampr, DspeE, pyrE+ This study pET21c+ Ampr Novagen pESE8 Ampr, for speE overexpression This study pESB8 Ampr, for speB overexpression This study 13

14 Table 2 Oligonucleotides used in this study Oligonucleotide Sequence1 speBKpnUp5 AAAGggtaccATGCGCCTCGTCTTCGGCGA speBHinUp3 CAGTaagcttGAAGGGGCTTGCGTGGGAGT speBEcoDw5 TCTTgaattcTACCGCCTCCTCACGGAGGG speBXbaDw3 CATAtctagaTGTGGTCCACCTCCCGGGAA speEup5kpn ggtaccTCACCAAGGACCAGCTCAAG speEup3hin aagcttAGGTCCTCCTTCTACTCGGC speEdw5xba tctagaAACTTTTTCCGGGGGGTGGGA speEdw3sac gagctcTCGTGGTAATCGTAGTAGCG sE5'NdeI GCGcatatgGACTACGGGATGTACTTCTTTGAGCACG sE3'Hind TaagcttTTGCCGGAGAGGCTACCCCTGAA sB5'NdeI GGTCACAcatatgCGCCTCGTCTTCGGCGAAAA sB3'Hind ATCGaagcttCTAAATGTGGTCCACCTCCCGGG 1 Lower case indicates restriction endonuclease recognition sites, KpnI, HindIII, EcoRI, XbaI and SacI. Downloaded from http://www.jbc.org/ by guest on September 11, 2016 14

15 Table 3 Effect of temperature on growth. Strain 70C 78C TTY1 1.08 0.74 MOSB 1.04 0.02 MOSBE 0.97 0.07 Turbidity was measured at 660 nm after culture for 5 days (MOSB) or 6 days (TTY1 and MOSBE). Downloaded from http://www.jbc.org/ by guest on September 11, 2016 15

16 Table 4. Purification of SpeE Total protein Specific activity Total activity Purification yield Step (mg) (nmol/min/mg) (mmol/min) (fold) (%) Cell-free extract 474.6 46.8 22.2 1 100 1st heat treatment 53.1 805.4 42.8 17 193 2nd heat treatment 52.3 836.9 43.8 18 197 Hiprep QXL 16.6 1738.9 28.8 38 130 Resource HIC Phe 9.7 2402.2 23.2 51 105 Aminopropyltransferase activity was performed at 60C, pH 8.5 for 20 min, with 1 mM spermidine as a substrate and 10 ng of protein per each reaction. Downloaded from http://www.jbc.org/ by guest on September 11, 2016 16

17 A B T. thermophilus genome TtspeB TtspeE PCR and clone into pBlueScriptSK+ PCR and clone into pBlueScriptSK+ XbaI SacI PCR and clone into pBHTK EcoRI XbaI PCR and clone into pBHTK pSED Downloaded from http://www.jbc.org/ by guest on September 11, 2016 pSBD KpnI HindIII KpnI HindIII HTK HTK pSBUKm pSEUKm Insert downstream Insert 3 half of region of TtspeE into TtspeB into pSBUKm pSEUKm EcoRI XbaI NdeI XbaI SacI HTK HTK pSBKm pSEKm Replace HTK region with NdeI EcoRI pyrE fragment of pINV pyrE pSEPE Fig. 1 Ohnuma et al. 17

18 . . . .10 . . . .20 . . . .30 . . . .40 . . . .50 . . . .60 Tth_HB8 1:..... MR L....... V F G EKDT P Y ...... EEA RV V VLP V P Y D LSL S F L P G A R R G P E A I L : 42 Tth_HB27 1:..... MR L....... V F G EKDA P Y ...... EEA RV V VLP V P Y D LSL S F L P G A R R G P E A I L : 42 Eco 1:MSTLGHQ Y DNSLVSNA F G FLRL P MNFQPYDS D A DW V I T GV P F D M AT S G R A G G R H G P A A I R : 60 Bsu 1:..... MR F DEAYSGK V F IASRPE W ...... EEA DA IL Y G M P M D W TV S Y RP G S R F G P SR I R : 49 Sto 1:MSDSR L L Y LNE.NSR L F A GFNK P T......SPF.. V II G L P L D IT S S FRP G S R F A P ST I R : 51 . . . .70 . . . .80 . . . .90 . . . 100 . . . 110 . . . 120 Tth_HB8 43:LA S RE LE P F L L E LG AAP... EEVG I HA A EP VP W VA G M AEES H R L I R E E A L R H L R A GK WVV : 99 Tth_HB27 43:LA S RE LE P F L L E LG AAP... EEVG I HA A EP VP W VA G M AEES H R L I R E E A L K H L R A GK WLV : 99 Eco 61:QV S TN L AWEHNRF P WNFDMR E R L N V V D C GD L V Y AF G D A R E MSEK L QAH A E K L L A A GK R ML :120 Bsu 50:EV S IG LE E Y SPY L DRDL...A DL NFF DAGD I P LPF G NP Q R S LD M I E E YVDSI L EK GK F P M :106 Sto 52:EY A QF I E F Y S I RT G IDMG... EVG FN D V GDV VMHPSDV EE NI R R I S D VTSYFAEK GK I II :108 . . . 130 . . . 140 . . . 150 . . . 160 . . . 170 . . . 180 Tth_HB8 100:AL GG D H S V T H PLV Q AH R EAL G D FS LLH V DAH A D LY P E W Q G SV YS H A S PFYR L LT EG F P .. :157 Tth_HB27 100:AL GG D H S V T H PLV Q AH R EAL G E FS LLH V DAH A D LY P E W Q G SV YS H A S PFYR L LT EG F P .. :157 Eco 121:SF GG D H F V T L PL LR AH AKHF G KMA L V H F DAH T D T Y A N .. G CE F D H GT M FY TAPK EG LIDP :178 Bsu 107:GM GG E H L V S W P VIK A MYKKY PD LA II H F DAH T D L RV DY E G EPL S H ST P IR K AAELIG P HN :166 Sto 109:GI GG E H S V T V G T V RG I K PDC.... V L S I DAH L D L RD E Y M G YK Y D H A CVMR R I SE Q G VK.. :162 . . . 190 . . . 200 . . . 210 . . . 220 . . . 230 . . . 240 Tth_HB8 157:.. L VQ V GI R A M D RD S L RL A R K K G V A L FP AH R I H RE..GL PL D EIL RA LG K R .. V Y ISL D F :211 Downloaded from http://www.jbc.org/ by guest on September 11, 2016 Tth_HB27 157:.. L VQ V GI R A M D RD S L RL A R K R G V A L FP AH R I H RE..GL PL D EIL EA LG K R .. V Y ISL D F :211 Eco 179:NHS VQ I GI R TEFD...... K D N G FT V LD A CQ V NDRSVDDV I A QV KQI V G DMP. V Y L T F D I :231 Bsu 166:.. V YSF GI R S GM KE EFEW A K E N G M H I SKFEV L E...... PL K E V L PK L A G R P. V Y V T I D I :217 Sto 162:.. IMEIA T R A V S KE E L DY A N KN G I A YLTP H Q I R LLGVRETAKK I V NNFRDCEK I Y V T Y D M :220 . . . 250 . . . 260 . . . 270 . . . 280 . . . 290 . . . 300 Tth_HB8 212:D A L DP S LM P S VG T P L PG G L S Y R Q V V DL L E AV F R . EK E VV G M D F VE L S P N G QF H A E M T A AQ :270 Tth_HB27 212:D A L DP S LM P S VG T P L PG G L S Y R Q V V DL L E AV F R . EK E VV G M D F VE L S P N G QF H A E M T A AQ :270 Eco 232:D C L DP A F A P G T G T P V I G G L T SDRA I K L V R GL KD..LN I V G M D V VE V A P . A Y D Q SE I T A LA :288 Bsu 218:D V L DP A H A P G T G T VDA G G IT S K E LL AS V HE I A R S E VN V K G A D L VE V A P .VY DHSE Q T A N. :275 Sto 221:D GI DP A Y A P GV A T P E P E G L DPTT V L D II SL I ID.. K R VV G F D V VE VS P .PH D P S G I T S VL :277 . . . 310 . . . 320 . Tth_HB8 271:LVYH AI G L K G L QAG WL S R E V DHI :293 Tth_HB27 271:LVYH AI G L K G L QAG WL S R E V DHI :293 Eco 289:AATL A L E M LY I QA A KKGE..... :306 Bsu 275:..TA S KI IR E M LL G FV K...... :290 Sto 278:G..AR I I L ETSAQI Y KA R S L ... :295 Fig. 2 Ohnuma et al. 18

19 Downloaded from http://www.jbc.org/ by guest on September 11, 2016 Fig. 3 Ohnuma et al. 19 30 20 Time (h) 10 0 10.00 1.00 0.10 0.01 A590

20 Spm Thm TTY1 (1/10) Spd Hcdp Agm Cdp Taa Put Fluorescent intensity MOSB (1/20) Unknown Y CadAgm X Polyamine A Put Downloaded from http://www.jbc.org/ by guest on September 11, 2016 Agm MOSBE (1/100) Cad Put 0 20 40 60 80 100 120 Retention time (min) Fig. 4 Ohnuma et al. 20

21 A 9.35 0.38 7.47 (3(3)4) 6.93 11.09 1.42 7.16 Downloaded from http://www.jbc.org/ by guest on September 11, 2016 5.05 (33) 5.57 (34) 10.59 2.48 13.47 B Time (min) C3 F7 OCHN H N NH N NHCOC 3F 7 COC3 F 7 N1-Aminopropylagmatine Aminopropylagmatine M-F 756 Relative abundance M-C3F7 606 M-COC3F7 578 (M 775) Fig. 5 Ohnuma et al. 21

22 O Cl O H O cHCl H NH2 O N O N H2N NaOH. aq N H O AcOH NH2 . HCl O O 1 2 3 O O 1) Br N CH3CN H2N . 2HBr O N N H O 2) 33% HBr 4 Downloaded from http://www.jbc.org/ by guest on September 11, 2016 H2 N H N N NH2 CH3 NH H . 5HCl H3 C N N 6 H2 N NH 5 Fig. 6 Ohnuma et al. 22

23 Hcdp Fluorescent intensity Spd Agm Taa Thm Hspd Spm 0 20 40 60 80 100 120 Retention time (min) Downloaded from http://www.jbc.org/ by guest on September 11, 2016 Fig. 7 Ohnuma et al. 23

24 A B kDa M 1 2 3 4 5 6 1 2 3 4 M kDa 97.4 66.2 97.4 45.0 66.2 31.0 45.0 21.5 31.0 14.4 C Synthesized Reaction product of TtSpeE Agmatine N1-aminopropylagmatine Fluorescent intensity a c e 0 min Agm Agm N1-aminopropyl- agm N1-aminopropyl- Downloaded from http://www.jbc.org/ by guest on September 11, 2016 agm Fluorescent intensity b d f 60 min Spd Spd Agm Agm N1-aminopropyl- agm 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 Retention time (min) Fig. 8 Ohnuma et al. 24

25 A B COOH COOH H2 N C H H2 N C H COOH H2 N C H HN HN NH NH H2N H2 N H2 N L -Ornithine L-Arginine L-Arginine NH2 CO 2 Arginine N N CO2 Arginine decarboxylase CH3 O decarboxylase Ornitine decarboxylase (speA ) N N HO C S (speA) ( speC) O CO2 H NH2 H2 N OH OH H2 N H N NH NH 2 SAM CO2 N NH2 NH Agmatine Agmatine SAM decarboxylase NH2 H 2O (speD ) N N NH2 CH3 O N N Agmatine N N HO C S CH3 O Urea ureahydorolase N N NH2 H2 N S (speB) O OH OH SAM CO 2 OH OH H2 N Polyamine agmatine NH2 Decarboxylated SAM aminopropyltransferase SAM decarboxylase Putrescine NH2 (speE) (speD ) N N NH2 N N N N H3C S CH3 O Downloaded from http://www.jbc.org/ by guest on September 11, 2016 N N H2 N S OH OH H2 N H O N MTA NH N NH 2 OH OH H Decarboxylated SAM Spermidine synthase 1 N -Aminopropylagmatine (speE) NH2 N N H 2O Aminopropylagmatine N N H3C S ureohydrolase O Urea (speB) MTA OH OH H2N H2N N NH2 N NH2 H H Spermidine Spermidine Long and branch ed polyamines C 1,3-Diaminopropane (Dap)* Putrescine (Put)* Cadaverine (Cad) NH2 H2N NH2 H2N H2N NH2 Norspermidine (Nspd)* Spermidine (Spd)* sym-Homospermidine (Hspd)* NH2 H 2N NH2 H2N N NH2 H2N N N H H H Thermine (Thm)* Spemrine (Spm)* Thermospermine (Tspm)* Homospermine (Hspm)* H H N NH2 NH2 N H2N N N NH2 H2N N H2N N N H2N N NH2 H H H H H H Caldopentamine (Cdp)* Thermopentamine (Tcdp)* Homocaldopentamine (Hcdp)* H N NH2 NH2 H2N N N N NH2 H2N N N H2N N N N H H H H H H H H Caldohexamine (Cdh)* Homocaldohexamine (Hcdh)* H2N N N N N NH2 H2N N N N NH2 N H H H H H H H H Tris(3-aminopropyl)amine (Mitsubisine, Mb)* N4-aminopropylspermidine Tetrakis(3-aminopropyl)ammonium (Taa)* NH2 NH2 H2N N H 2N N+ NH2 N H2N NH2 H2N NH2 H2N Fig. 9 Ohnuma et al. 25

26 N1-aminopropylagmatine: A new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, thermus thermophilus Mio Ohnuma, Yusuke Terui, Masatada Tamakoshi, Hidemichi Mitome, Masaru Niitsu, Keijiro Samejima, Etsuko Kawashima and Tairo Oshima J. Biol. Chem. published online June 27, 2005 Access the most updated version of this article at doi: 10.1074/jbc.M413332200 Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at Downloaded from http://www.jbc.org/ by guest on September 11, 2016 http://www.jbc.org/content/early/2005/06/27/jbc.M413332200.citation.full.html#ref-list-1

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