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Home , Agmatine deiminase, Glutamic acid

Biosci. Biotechnol. Biochem., 72 (2), 445–455, 2008

Occurrence of Pathway for Synthesis in Selenomonas ruminatium

Shaofu LIAO,1;* Phuntip POONPAIROJ,1;** Kyong-Cheol KO,1;*** Yumiko TAKATUSKA,1;**** y Yoshihiro YAMAGUCHI,1;***** Naoki ABE,1 Jun KANEKO,1 and Yoshiyuki KAMIO2;

1Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiyamachi, Aobaku, Sendai 981-8555, Japan 2Department of Human Health and , Graduate School of Comprehensive Human Sciences, Shokei Gakuin University, 4-10-1 Yurigaoka, Natori 981-1295, Japan

Received August 28, 2007; Accepted November 16, 2007; Online Publication, February 7, 2008 [doi:10.1271/bbb.70550]

Selenomonas ruminantium synthesizes and Polyamines such as putrescine, cadaverine, and putrescine from L- and L- as the essential are essential constituents of peptidoglycan constituents of its peptidoglycan by a constitutive lysine/ and they play a significant role in the maintenance of the (LDC/ODC). S. ruminantium integrity of the cell envelope in Selenomonas ruminan- grew normally in the presence of the specific inhibitor tium, Veillonella parvulla, V. alcalescens, and Anaero- for LDC/ODC, DL- -difluoromethylornithine, when vibrio lipolytica.1–3) When S. ruminantium and two was supplied in the medium. In this study, species of Veillonella are grown in a medium supple- we discovered the presence of arginine decarboxylase mented with putrescine or cadaverine, putrescine and (ADC), the key in agmatine pathway for cadaverine respectively link covalently to the -carbox- putrescine synthesis, in S. ruminantium. We purified yl group of the D-glutamic residue of the peptido- and characterized ADC and cloned its gene (adc) from glycan, which is catalyzed by diamine: intermediate S. ruminantium chromosomal DNA. ADC showed more , in normal cell growth.3–7) In S. ruminanti- than 60% identity with those of LDC/ODC/ADCs from um, both putrescine and cadaverine are constitutively Gram-positive , but no similarity to that from synthesized from L-ornithine and L-lysine by lysine/ Gram-negative bacteria. In this study, we also cloned ornithine decarboxylase (LDC/ODC), which has decar- the aguA and aguB genes, agmatine deiminase boxylase activities towards both L-lysine and L-ornithine 8) (AguA) and N-carbamoyl-putrescine with similar Km. Hence, our LDC was designated (AguB), both of which are involved in conversion from lysine/ornithine decarbozylase (LDC/ODC).9) LDC/ agmatine into putrescine. AguA and AguB were ex- ODC activities are prevented irreversibly by DL-- pressed in S. ruminantium. Hence, we concluded that difluoromethyllysine (DFML) or DL--difluoromethylor- S. ruminantium has both ornithine and agmatine path- nithine (DFMO).8) Since S. ruminantium has no L- ways for the synthesis of putrescine. ornithine specific decarboxylase,10) LDC/ODC is thought to be an important enzyme supplying putrescine Key words: Selenomonas ruminantium; agmatine path- and cadaverine in the construction of diamine-contain- way for putrescine synthesis; arginine de- ing peptidoglycan.8) In S. ruminantium, the production carboxylase; agmatine deiminase; N-carba- of LDC/ODC is highly regulated and strictly linked to moyl-putrescine amidohydrolase the growth phase, i.e., a drastic decrease in LDC/ODC

The first and the second authors contributed equally. y To whom correspondence should be addressed. Tel/Fax: +81-22-381-3347; E-mail: [email protected] * Present address: Department of Science and Nutrition, Meiho Institute of Technology, 23 Ping Kuang Rd., Nei Pu Pingtung, 91212, Taiwan ** Present address: Biotech Central Research Unit, National Center for Genetic Engineering and Biotechnology, 113 Paholyothin Rd., Klong 1, Klong Luang, Pathumthani, 12120, Thailand *** Present address: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong-dong, Jeongeup-shi, Jeolabuk-do, 580-185, Republic of Korea **** Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA ***** Present address: Department of , Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA Abbreviations: LDC, L-lysine decarboxylase; ODC, L-ornithine decarboxylase; ADC, L-arginine decarboxylase; AguA, agmatine deiminase; AguB, N-carbamoyl-putrescine amidohydrolase; NCP, N-carbamoyl-putrescine; DFML, DL--difluoromethyllysine; DFMO, DL--difluoromethyl-ornithine; DFMA, DL--difluoromethylarginine; PALP, pyridoxal-50-phosphate 446 S. LIAO et al. activities occurs on entry into the stationary phase of cell vant was from Wako Pure Chemical Industry (Osaka, growth, due to rapid degradation of LDC/ODC.8) Rapid Japan). Anti-rabbit and anti-mouse IgG (Fc)-alkaline degradation of LDC/ODC caused a drastic decrease in phosphatase conjugate were from Promega (Madison, the intracellular free cadaverine content in the stationary WI). ABI PRIZM BigDye Terminator Cycle Sequencing phase, whereas the putrescine, spermidine, and Ready Reaction kit was from Applied Biosystems contents in the stationary phase were on a level with that (Foster city, CA). Unless otherwise stated, chemicals in the exponential phase (unpublished data). In the used in this study were of the best grade commercially current study, we isolated a new of 22 kDa (P22), available. which is induced on entry into the stationary phase of putrescine-grown S. ruminantium cells, as a regulatory Bacterial strains and plasmids, and culture condi- factor in LDC/ODC degradation by the ATP-dependent tions. The strains used in this study included S. rumi- system.11) In the preceding study, we nantium subsp. lactilytica,9) and E. coli DH5 and E. coli characterized P22 and found that it is a direct counter- Rosetta (DE3) (Novagen) which were used as host part of eukaryotic antizyme (AZ) in the acceleration of strains. Culture media included a Bacto-tryptone (1%)- the degradation of S. ruminantium LDC/ODC due to its Bacto- extract (1%)-glucose (0.5%) medium sup- binding to the P22- in the LDC/ODC plemented with 0.01% caproic acid (TYG) and CD molecule.9,12) We also found that P22 is a ribosomal medium10) for S. ruminantium, and an LB medium for protein of S. ruminantium with no similarity to mouse E. coli strains. S. ruminantium was grown under anae- AZ in sequence.12) Recently, we observed robic conditions, as described previously.9) that S. ruminantium grew normally even in the presence of 5 mM DFMO when 10 mML-arginine was supplied in Reaction mixture and enzyme assay. ADC, AguA, and a chemically defined medium (CD medium).9) When AguB were assayed by measuring agmatine, NCP, and S. ruminantium was grown with 14C-labeled L-arginine putrescine, which were converted from L-arginine, in the presence of DMFO, the radioactive L-arginine was agmatine, and NCP respectively by ADC, AguA, and incorporated into the cells and converted into agmatine AguB. For ADC assay, the reaction mixture contained and putrescine. These findings suggest that S. ruminan- 50 mM potassium phosphate buffer (pH 6.5), 10 mML- tium has an agmatine pathway besides the ornithine arginine, 50 mM PALP, 1 mM dithiothreitol (DTT), and pathway for the synthesis of putrescine. We discovered an enzyme peparation in a total volume of 100 ml. AguA the presence of L-arginine decarboxylase (ADC), the and AguB activities were assayed in 50 mM PIPES first enzyme in the agmatine pathway. We purified and buffer (pH 6.5) containing 1 mM DTT, 25 mM agmatine characterized ADC, and cloned the ADC gene (adc) for AguA and 10 mM NCP for AguB, and the enzyme from S. ruminantium chromosomal DNA. Furthermore, preparation in a total volume of 100 ml. The reaction we identified agmatine deiminase (aguA) and N-carba- mixtures were incubated for 10 min at 50 C for ADC moylputrescine (NCP) amidohydrolase (aguB) genes and 40 C for AguA and AguB. The reaction was from a flanking region of adc. They are involved in the stopped by adding TCA at a final concentration of 5%, conversion of agmatine to putrescine. These genes were and the precipitate was removed by centrifugation at cloned and expressed in Escherichia coli, and recombi- 10;000 g for 5 min. The amounts of agmatine, NCP, nant AguA and AguB (rAguA and rAguB) were purified and putrescine were measured by HPLC using a TSKgel and characterized. Expression of AguA and AguB in Polyaminepak column (Tosoh, Tokyo) and cellulose S. ruminantium was confirmed immunologically using thin-layer electrophoresis using a cellulose plate 5716 anti-rAguA and -rAguB antisera, respectively. (Merk, Darmstadt, Germany) by the method described previously.14) One katal (kat) of each enzyme activity Materials and Methods was defined as the formation of 1 mole each of agmatine, NCP, and putrescine per second. Specific Materials. DFML, DFMO, and DL--difluorometh- activity is given as kat/kg of enzyme. The kinetic ylarginine (DFMA) were kind gifts from Merrel Daw parameters, Km,Vmax, and Ki were calculated from Research Institute (Cincinnati, OH). Agmatine was initial rate measurements for the of purchased from Aldrich (St. Louis, MO). NCP was L-arginine, the formation of NCP, and putrescine by synthesized chemically according to the method describ- fitting to the Michaelis-Menten equation using a non- ed by Smith and Garraway.13) Plasmids pMW118 and linear regression algorithm. pMW119 were purchased from Nippon gene (Tokyo) and plasmids pET15b and pET28b were from Novagen Purification of S. ruminantium ADC. S. ruminantium (Madison, WI). Restriction , T4 DNA , cells were grown in 100 liters of TYG medium and Taq DNA polymerase were from Takara, (Otsu, supplemented with 20 mML-arginine. Cells were har- Japan). A HiTrap chelating HP column, Hybond ECL vested at mid-exponential phase by continuous centri- membrane, and L-[U-14C]arginine (11 GBq/mmol) were fugation and washed once with 100 mM potassium from GE Healthcare Bio-Science (Chalfont St. Giles, phosphate buffer (pH 7.0) containing 5 mM MgCl2. UK). Lysylendopeptidase and Freund’s complete adju- The cells (wet weight, 134.7 g) were suspended in Agmatine Pathway for Putrescine Synthesis in S. ruminantium 447

300 ml of 50 mM potassium phosphate buffer, pH 7.0, sequencing of adc, the amino acid sequences corre- containing 10 mM 2-mercaptoethanol and 0.1 mM PALP sponding to the N-terminal 27-residue and 23-, 7-, 11-, (buffer A), and disrupted in a French pressure cell (SLM and 10-residue segments were found. Instrument, Rochester, NY) at 1,000 kg/cm2. After centrifugation at 20;000 g for 20 min, the supernatant Preparation of oligonucleotide primers for cloning of obtained was dialyzed against 250 mM potassium phos- adc from S. rumiantium. According to the N-terminal phate buffer (pH 7.0) containing 10 mM 2-mercapto- (Met1-Asp-Arg-Ile-Asn-Gln-His7) amino acid sequence ethanol and 0.1 mM PALP, and heated at 62 C for of intact ADC and its lysylendopeptidase fragment, 10 min with gentle swirling. After centrifugation at NH2-Asp-Glu-Ala-His-Gly, the oligonucleotide primers 20;000 g for 30 min, the supernatant was collected and Fw (50-ATGGA(TC)CG(ATGC)AT(TCA)AA(TC)CA- then put on a TSKgel DEAE-Toyopearl 650M column (AG)CA-30) and Rv (50-GT(ATGC)CC(AG)TG(ATG- (40 240 mm; Tosoh, Tokyo), which was equilibrated C)GC(TC)TC(AC)TC-30) were designed. In combina- with 50 mM potassium phosphate buffer, pH 7.0 (buf- tion with each primer, an approximately. 600-bp frag- fer B). The column was washed with buffer B and then ment was amplified from the chromosomal DNA of eluted with a linear gradient of NaCl in buffer B (0 to S. ruminantium by PCR using a Takara Ex Taq 500 mM). Active fractions, eluted from 250 to 350 mM polymerase. The amino acid sequence predicted from NaCl, were collected and dialyzed against buffer A, the DNA sequence in the amplified DNA fragment and then put on a TSKgel DEAE-5PW column (21:5 corresponded to that of the 16-, 23-, 7-, 11-, and 4- 150 mm; Tosoh, Tokyo), which was equilibrated with residue segments H7TAPVYEAMLELRKRR22,E71AEE- buffer B. The column was washed with buffer B and LTADAFGAQHAFFMVHGTT92,V99LSTVRA105,A149- eluted with a linear gradient of NaCl (0 to 400 mM). The LSDVERAIRQ159, and K193VLV196 respectively, of active fractions eluted from 220 to 250 mM NaCl in intact ADC. Hence, the 600-bp PCR fragment was buffer B were pooled. After it was dialyzed against used as a DNA probe in the cloning of adc from the buffer A, the enzyme fraction was put on a TSKgel HA- S. ruminantium chromosomal DNA. 1000 column (7:5 75 mm; Tosoh) equilibrated with buffer B. The column was washed with buffer B. The Cloning of S. ruminantium ADC gene (adc) and its enzyme was then eluted with a linear gradient of DNA sequencing. A 3.5-kbp EcoRI fragment of chro- potassium phosphate from 50 to 200 mM, pH 7.0. The mosomal DNA of S. ruminantium was strongly hybri- fractions eluted from 60 to 80 mM potassium phosphate dized with the probe. We amplified the 3.5-kb fragment were pooled. After dialysis against buffer A, the enzyme by inverse PCR using internal primers of a 605-bp fraction was put on a Mono Q HR 5/5 column fragment. As a result of inverse PCR walking, the (5 50 mm; Pharmacia, Uppsala) equilibrated with nucleotide sequence of a 10.5-kb region containing adc buffer B. The column was washed with buffer B and and its flanking region was determined. eluted with a liner gradient of NaCl in buffer B (0 to 500 mM). The fractions eluted from 280 to 330 mM NaCl ORF identification, homology search, and alignment were pooled and dialyzed against buffer A. of multiple nucleotide and amino acid sequences. Protein and nucleotide sequences were compared with Determination of the N-terminal and internal amino those on databases using FASTA and BLAST programs acid sequences of S. ruminantium ADC preparation. implemented at the EMBL/GenBank/DDBJ nucleotide The purified ADC preparation (200 mg) was dissolved in sequence databases and the SWISSPROT/NBRF-PIR 1 ml of 70% formic acid containing 400 mg of CNBr and protein sequence databases. Multiple-sequence align- kept in the dark under N2 gas at 24 C for 24 h. ment was done using a GENETYX program (Software Alternatively, the purified ADC preparation (100 mg) Development, Tokyo). was completely digested with 1 mg of lysylendopepti- dase at 37 C for 12 h in 0.4 ml of 100 mM Tris–HCl Construction of plasmids pET28b-adc, -orf6, and buffer (pH 9.0). fragments were subjected to -orf7 for expression of S. ruminantium ADC, ORFs 6 SDS–PAGE in a Tris-tricine buffer system, and then the and 7 in E. coli. To construct an expression system for separated were transferred to an Immobilon-P rADC, ORFs-6 and -7, DNA fragments, including a membrane (Millipore, Bedford, MA) by the method cording region of ADC, ORFs-6 and -7, were amplified described previously.14) The N-terminal amino acid by PCR and ligated into plasmid pET28b. The resulting sequences of the peptides were analyzed with an plasmids were designated pET28b-adc, -orf6, and -orf7. Applied Biosystems protein sequencer Model 491. The Each plasmid was transformed into E. coli Rosetta N-terminal sequence of the purified ADC was deter- (DE3). Cells carrying each plasmid was grown in LB mined to be M1DRINQHTAPVYEAMLELRKRRVV- medium containing kanamycin (20 mg/ml) at 37 C with PFD27. The amino acid sequences of four internal shaking to an optical density of 0.6 at 600 nm, IPTG was peptide fragments, that were isolated were determined to added at a final concentration of 0.4 mM, followed by be EAEELTADAFGAQHAFFMVHGTT, VLSTVRA, incubation with shaking for an additional 2 h. The cells ALSDVERAIRQ, and KVLVDEAHGT. From the DNA were collected by centrifugation at 4 C and suspended 448 S. LIAO et al. in 50 mM potassium phosphate buffer (pH 7.4) contain- Putrescine ing 50 mM PALP, and disrupted in a French pressure cell. Agmatine The cell lysate was centrifuged at 8;000 g for 20 min at 4 C, and the supernatant was used for purification of Arginine rADC and ORFs 6 and 7.

Site-directed mutagenesis of adc and orfs 6 and 7. Site directed mutagenesis was done by the overlapping Agm 0 2 4 6 8 10 12 24 15,16) extension method. The mutation of each mutant was Time (h) confirmed by DNA sequencing. Fig. 1. Conversion of 14C-Arginine into Agmatine and Putrescine in Purification of the recombinant . Crude S. ruminatium. recombinant ADC and ORFs 6 and 7 with His-Tag at S. ruminantium cells were cultured in CD medium supplemented with 10 mML-[U-14C]arginine (0.18 MBq) and 5 mM DFMO. At the the C-terminal were purified with a HiTrap chelating HP times indicated, cells were harvested and extracted with 5% TCA. column using a linear gradient from 0 to 500 mM After the precipitate was discarded by centrifugation at 10;000 g, imidazole in 20 mM sodium phosphate, pH 7.2, contain- the supernatant was analyzed by cellulose thin-layer electrophoresis 14 ing 500 mM NaCl. ADC and ORFs 6, and 7 were eluted to detect C-arginine, -agmatine, and -putresine. in a fraction containing 150 mM imidazole. The active fractions were collected and dialyzed against 50 mM Results and Discussion PIPES buffer, pH 7.0, containing 1 mM DTT (buffer C) and then applied to a DEAE-5PW column equilibrated Conversion of L-arginine into agmatine and putres- with buffer C (Tosoh, Tokyo). After washing of column cine with the buffer C, the enzymes were eluted with a linear S. ruminantium cells were grown at 37 CinCD gradient of NaCl from 0 to 500 mM in buffer C. The medium supplemented with 10 mM 14C-labeled L-argi- active fractions from the DEAE-5PW column was nine in the presence of 5 mM DFMO. At the times dialyzed against buffer C and then sulfate indicated in Fig. 1, cells were harvested and treated with was added at a final concentration of 1,000 mM and 10% cold TCA. After centrifugation, the TCA soluble applied to a phenyl-5PW column (Tosoh), which was fraction was analyzed by cellulose thin-layer electro- equilibrated with buffer C containing 1,000 mM ammo- phoresis (Fig. 1). As shown, the labeled L-arginine was nium sulfate. After the column was washed with incorporated into the cells up to 4-h after incubation. buffer C containing 1,000 mM ammonium sulfate, en- Thereafter, the amount of L-arginine decreased between zyme fractions were eluted with a linear gradient of 0.5 and 8, accompanied by an increase in agmatine and ammonium sulfate from 1,000 mM to 0 mM in buffer C. putrescine. After 10 h of incubation, no L-arginine was The molecular masses of the recombinant proteins were detected in the TCA soluble fraction. On the other hand, measured with a TSK gel G3000SW gel filtration the accumulation of putrescine in the TCA soluble column (Tosoh; diameter, 0.75 cm; height, 30 cm) fraction proceeded up to 12 h after incubation. The data equilibrated with 50 mM potassium phosphate buffer clearly showed that 14C-lableled L-arginene incorporated (pH 6.0) containing 200 mM NaCl, using albumin (64.7 into the cells was converted into agmatine and putres- kDa), ovalbumin (45.8 kDa), chymotrypsinogen (19.9 cine in S. ruminantium in the presence of 5 mM DMFO. kDa), and ribonuclease A (15.4 kDa) as molecular Therefore, it is suggested that S. ruminantium has ADC standards. pathway for the synthesis of putrescine.

Preparation of anti-S. ruminantium ADC, -rAguA, Detection of ADC in the crude extract from S. rumi- and -rAguB antisera. Mice antiserum raised against nantium rADC, rAguA, and rAguB was prepared by the method The ADC activity during growth of the S. ruminan- described previously.8) tium cells in TYG medium supplemented with 10 mM L-arginine and 0.01% capric acid was examined. Miscellaneous. Quantitative assay of protein and Sonicated extracts from the cells were taken at various SDS–PAGE were performed by the methods described times after inoculation and ADC activity was assayed. previously.8) ADC activity was detected, and the activity in all the cells in culture increased up to 6 h, and reached ap- Nucleotide sequence accession numbers. The nucleo- proximately 3:9 109 kat/liter of culture, and decreas- tide sequences of adc, orfs-1, -2, -3, -4, -5, -6, and -7 ed thereafter. Accordingly, the timing of cell harvest for have been deposited in the DDBJ/EMBL/GenBank preparation of crude extract from the cells was taken to nucleotide sequence databases under accession Nos. be during the period, 3.5–4.0 h (O.D.660 = 1.0) after AB198397, AB198398, AB196518, AB198392, AB198- inoculation. When the cells were grown in CD medium 393, AB198394, AB198395, and AB198396. without L-arginine, no detectable ADC activity was found in the crude extract prepared from the cells. Agmatine Pathway for Putrescine Synthesis in S. ruminantium 449 Table 1. Purification of S. ruminantium ADC

Total volume Total protein Total activity Specific activity Yield Purification Purification step (ml) (mg) (109 kat) (103 kat/kg) (%) (fold) Crude extract 318 3200 339 0.123 100 1 Heat treatment 310 2310 425 0.184 108 1.5 DEAE-Toyopearl 650M 232 331 132 0.399 33.5 3.25 DEAE-5PW 25 19.7 12.5 0.635 3.2 5.16 HA-1000 21 0.42 7.18 17.1 1.8 139 MonoQ HR 5/5 6.3 0.117 29.1 249 7.4 2024

Purification and characteristics of S. ruminantium by the ADC preparation with Ki values of 0.8 mM and ADC 2.1 mM respectively (Fig. 2A and B). In the range from 0 Purification to 0.2 mM DFMA, plots of the reciprocals of reaction Through the five purification steps shown in Table 1, velocities against L-arginine and L-lysine gave a series of ADC was purified about 2,030-fold to electrophoretic straight lines intercepting a single point on the 1/v axis, homogeneity with a specific activity of 0.25 kat/kg of indicating that the inhibition type of DFMA is compet- protein. At the final step of purification, total specific itive and that the catalytic domains of ADC towards ADC activity increased to about 15 times that at the the two substrates are identical. When the enzyme was semi-final step (HA-1000 column chromatography). incubated for 60 min at 4 C with DFMA (10 mM) and After MonoQ HR 5/5 chromatography, some contam- dialyzed against buffer A, the decarboxylating activities inants, which prevented the ADC activity, have been towards both substrates were irreversibly lost (data not removed. Though the inhibitors of ADC were not shown), indicating that DFMA is an irreversible inhibitor identified, it is assumed that the inhibitors are entity with of S. ruminanitum ADC. On the other hand, neither high molecular mass, because they did not pass through DFML nor DFMO affected ADC activities. a cellulose dialyzing bag. The N-terminal amino acid Optimal conditions for enzyme activity sequence of the enzyme was M1DRINQHTAP10VY- The optimal pH for enzyme activity was 6.5, same as EAMLELRKRR22. The purified ADC can be stored at that for an E. coli inducible ADC, but not for constit- 80 C for at least 6 months without any appreciable utive ADC. At pH 7.0, 8.0, and 9.0, the activity de- loss in the presence of 1 mM PALP. creased to 80%, 70%, and 50% as compared to that at The presence of the enzyme molecule in dimeric form pH 6.5, respectively. The optimal temperature for the with ADC activity enzyme activity was estimated to be 60 C. The enzyme The apparent molecular mass of the purified ADC activities at 10 min of incubation at 50, 55, 60, and 70 C preparation was 120 kDa as judged by gel filtration were about 4, 7, 10, and 1.2 times higher respectively analysis, and was 58 kDa on SDS–PAGE (Table 2). On than at 40 C. gel filtration, the monomeric form of ADC of 58 kDa Enzyme stability was also separated with no enzyme activity (data not The purified enzyme preparation, which was stored at shown). The data show that the active ADC comprised pH 6.0 to 7.5 at 4 C, maintained activity for at least 1 two identical monomeric subunits. month without loss. Full enzyme activities and approx- specificity and inhibitor imately 50% of them were retained at 50 and 60 C To determine the substrate specificity of the enzyme respectively for 60 min in the presence of 1 mM PALP. preparation, the enzyme concentrations used in the On the other hand, at 50 C in the absence of PALP, enzyme reaction were held at about 1.0–100 nM. L- 90% of the activity was lost within 10 min. No enzyme Arginine was the preferred substrate for this enzyme, activity was observed in the enzyme preparation heated whereas L-lysine was attacked at a rate of 10% of that at 70 C for 60 min even in the presence of 1 mM PALP. for L-arginine. Neither D-lysine, L-ornithine, nor L- Comparison of properties of S. ruminantium ADC acted as substrate. The kinetics of decarboxyl- with other ADCs ation of L-arginine and L-lysine by the ADC preparation Table 2 shows a comparison of properties of S. ru- were analyzed by measuring initial velocities over the minantium ADC with that of ADCs from E. coli,17–19) range of substrate concentrations (1 to 8 mM). Over the ,20) and rat.21) S. ruminantium ADC was mark- concentration range used, both L-arginine and L-lysine edly different from the other ADCs listed in this table in behaved as Michaelis-Menten-type substrates (Fig. 2A enzyme properties, except for a similarity in the and B). A double-reciprocal plot of the initial velocities requirement for the enzyme activity. S. ruminantium demonstrated that the Km values for L-arginine and ADC of 120 kDa, consisted on two identical subunits of L-lysine were 5.6 mM and 50 mM respectively, and that 58 kDa, whereas the E. coli constitutive (296 kDa) and 1 1 the Vmax values were 12.5 and 5.9 mmol min mg of inducible (820 kDa) ADCs consisted of three identical protein for L-arginine and L-lysine respectively. DMFA subunits of 72 kDa18) and 10 identical subunits of inhibited the decarboxylation of L-arginine and L-lysine 820 kDa22) respectively. It is also of interest that 450 S. LIAO et al. Table 2. Comparison of S. ruminantium ADC with ADCs from E. coli, Plant, and Rat

E. coli Plant Property S. ruminantium Rat Inducible (AdiA) Constitutive (SpeA) ( max) Decarboxylase type type I type I type III type III type III Subunit Mr 58,000 82,000 72,000 74,000 55,000 120,000 820,000 296,000 240,000 — Native M r (Dimer) (Decamer) (Tetramer) (Trimer) Cofactor PLP PLP PLP PLP PLP Substrate specificity 0.03 Km L-arginine (mM) 5.6 0.065 0.0461 0.75 (with Mg2þ) Km L-ornitine (mM) — — — — 0.25 Km L-lysine (mM)50— — — — L--Chloro-- Inhibitor DFMA Putrescine, Spermidine Agmatine, Putrescine Ca2þ,Co2þ guanidovaleric acid K for ADC activity i 0.8 0.026 — — — (mM) K for L-LDC activity i 2.1 — — — — (mM) Optimal pH 6.5 5.2 8.4 — 8.3 Optimal temperature 60 37–40 — — 25 (C) pH stability 6.5–7.5 (>80%) — — — — Thermal stability for 30 min With PLP 60 C (54%) — — 30–50 C— Without PLP 35 C (50%) — — — —

A B

0.1 mM DFMA 0.2 mM DFMA 0.05 mM DFMA 0.1 mM DFMA 1.0 1.0 mg protein) mg protein) • • No inhibitor

No inhibitor µ 0.5 µ 0.5 1/v (1/ mol/min 1/v (1/ mol/min

0 1.0 0 0.1 -1 1/[L-arginine] (mM ) 1/[L-lysine] (mM-1)

Fig. 2. Lineweaver-Burk Plots of L-Arginine Decarboxylase (A) and L-Lysine Decarboxylase (B) Activities of the Purified S. ruminantium ADC Preparation with and without DFMA.

S. ruminantium ADC activity was not inhibited by gene and 7 ORFs (Fig. 3A). The ORF for the adc gene putrescine, spermidine, agmatine, or -hydroxy--gua- (solid arrow in Fig. 3A) encoded a 485-amino acid nidovaleric acid, which are known to be strong protein of 53,313 Da whose N-terminal sequence match- inhibitors of the other ADCs shown in Table 2.23,24) A ed that determined for purified ADC. The adc gene, comparison of the fold type of S. ruminantium ADC and spanning positions 2,961–4,410 within the cloned others is discussed below. genomic sequence, started with an ATG codon and ended with a TAA stop codon. Putative 35 (TTGCTT) Nucleotide sequence of the S. ruminantium ADC gene and 10 (TAAAAT) sequences were found 84 and (adc) 61 bp upstream of the translation initiation codon of adc The determined 11.5-kbp sequence included the adc respectively (Fig. 3B). Ten base pairs upstream of the Agmatine Pathway for Putrescine Synthesis in S. ruminantium 451

0 5 10 kbp

orf1 2 adc orf3 orf4 orf5 orf6 orf7

Fig. 3. Genetic Organization of adc and Its Flanking Regions, and Nucleotide Sequence of the adc of S. ruminantium. A, Genetic organization of a 10.5-kbp fragment from S. ruminantium chromosomal DNA, including the adc and its flanking regions. The solid and open arrows show the adc gene and seven other predicted ORFs and the directions of their transcription. B, Nucleotide sequence of the adc. The strand shown is in the 50 to 30 direction, and numbers of nucleotides were counted from the first nucleotide of the 10.5-kbp fragment. The deduced amino acid sequence is indicated by the single-letter code under the nucleotide sequence. The amino acid sequence determined chemically, putative ribosomal binding site, predicted promoter sequence, and translation termination codon are indicated by single underlines, double underline, black-shaded boxes, and a single asterisk respectively.

ATG codon, a ribosomal binding site consensus se- even in the presence of PALP. On the other hand, quence (GGAGG), was found at position 2,951–2,955. S. ruminantium ADC showed no homology with an No termination loop was found downstream of the TGA E. coli constitutive ADC,28) which belongs to fold stop codon. type III as do eukaryotic ODC and plant ADC.25) The To confirm that adc was the gene encoding S. rumi- data suggested that S. ruminanitum ADC belongs to fold nantium ADC, the cloned adc was expressed in E. coli. type I decarboxylase. The recombinant ADC (rADC) was purified and its characteristics were compared with that of ADC Nucleotide sequencing analysis of the flanking region preparation. The purified rADC protein had character- of adc istics identical to those of the native ADC protein. Two orfs(orf6 and orf7 in Fig. 3A) that showed high homology with the genes encoding agmatine utilization Amino acid sequence homologies of S. ruminantium enzymes, were found downstream of the adc gene. ADC with other bacterial decarboxylases ORF6, which consists of 372 amino , had 54% and A comparison of the deduced amino acid sequence 43% identity in amino acid sequence with agmatine of S. ruminantium ADC with those of other possible deiminase of Peudomomas aeruginosa29) and Strepto- bacterial decarboxylases revealed that S. ruminantium coccus mutans30) respectively, and 48% identity with ADC showed 40 to 68% identities with possible peptidyl-arginine deiminase-like protein of Listeria arginine, ornithine, and lysine decarboxylases from monocytogenes (accession no. AAG03681). ORF7, genome sequencing of Streptococcus pneumoniae, Clos- which is composed of 292 amino acids, had 57% tridium perfringens, Fusobacterium nucleatum, Bacillus identity in amino acid sequence with NCP amidohy- cereus, and putative decarboxylases from Symbiobacte- drolase of Peudomomas aeruginosa. ORF7 also showed rium thermophilium and Thermoanaerobacter tengcon- 71, 58, 57, and 55% identities with - gesis (Fig. 4). Grishin et al. have proposed a topo- family proteins from Streptococcus pneumo- graphic model of the PALP-binding domain of niae (accession no. AAK75046), Erwinia carotovora eukaryotic and prokaryotic ODCs by the analysis of (accession no. CAG77171), Caulobacter crescentus known barrel structures of ODCs.25) They classified the (accession no. AAK22199), and Burkholderia pseudo- PALP-utilizing enzymes into seven fold types from the mallei (accession no. CHA34094) respectively. These predicted secondary structures, and showed that the results suggest that S. ruminantium has an agmatine prokaryotic ODCs, prokaryotic LDCs, and bacterial pathway for putrescine synthesis that includes ADC, inducible ADCs belong to fold type I. One of these, agmatine deiminase, and NCP amidohydrolase. inducible ODC from Lactobacillus 30a, has three amino ORFs-1, -2, -3, -4, and -5, encoding 467-, 207-, 286-, acids involved in PALP binding in its active center. Lys- 439-, and 391-amino acid proteins respectively, were 355 in the Ser-X-His-Lys motif, which is commonly also identified. The amino acid sequences of ORFs-1, -2, found in PALP-dependent decarboxylase, forms a Schiff -3, -4, and -5 exhibited 61, 31, 67, 59, and 61% overall base with PALP. An invariant Asp-316 residue is also a identify with putative Naþ/melibiose symport or related common motif found in many PALP-dependent en- transporter (accession no. ZP 00732149), putative cyti- zymes.25,26) Although S. ruminanitum ADC showed less dylate kinase (accession No. EAA23705), putative than 20% identity in amino acid sequence of inducible spermidine synthase (accession no. AAK75042), puta- ODC from Lactobacillus 30a, E. coli constitutive LDC, tive (accession no. AAB37- and E. coli inducible ADC (AdiA),27) which belongs to 373), and putative carboxynorspermidine decarboxylase fold type I (Table 2), S. ruminanitum ADC and pre- of Streptococcus pneumoniae (accession No. BAB07- dicted enzymes listed in Fig. 4 shared three residues 677) respectively. At positions 10 to 15 bp upstream of (His-117, Asp-197, and Lys-228) that are involved in the first ATG codons of orfs4, -5, -6, and -7, putative PALP binding in fold type I decarboxylases (Fig. 4). An ribosomal binding sites were observed. An inverted- H117A mutant of S. ruminantium ADC lost its activity repeat sequence consisting of a stem-loop with a 12-bp 452 S. LIAO et al.

Fig. 4. Sequence Data of the Predicted ADC/ODC/LDC from Genome Sequencing. The sequence data were obtained from GenBank (http://www.ncbi.nlm.nih.gov): S. pneumoniae (Spn) LDC (accession No. AAK75040), Cl. perfringens (Cpe) LDC (accession no. BAB80255), F. nucleatum (Fnu) ODC (accession no. EAA24580), S. thermophilum (Sta) ODC (accession no. BAD42250), T. tengcongesis (Ttn) LDC (accession no. AAM24281), B. cereus (Bce) ADC (accession no. AAP10882), E. coli (Eco) constitutive LDC (accession no. BAA21656), E. coli inducible ADC (AdiA) (accession no. AAA97017), and Lactobacillus 30a (L30) ODC (accession no. AAA64830). Agmatine Pathway for Putrescine Synthesis in S. ruminantium 453 A Control ORF6 ORF7 reaction reaction

Putrescine - Agmatine - NCP - Put NCP 0 min 30 min 0 min 30 min B Agm

ORF6 ORF7 H2NCNH NH H2NCNHNH2 H2N NH2

NH2 H2O NH3 O CO2+NH3 Agmatine NCP Putrescine

Fig. 5. Cellulose Thin-Layer Electrophoresis of the Reaction Products from Agmatine and NCP by the Recombinant ORF-6 and -7 (A), and a Schematic Representation of the Enzyme Reactions by ORF-6 and -7 (B). Thin-layer electrophoresis was done as described in ‘‘Materials and Methods.’’ The reaction products were detected by spraying with ninhydrin reagent.2) arm appeared at positions 290 bp to 321 bp downstream of the stop codon of orf7. 10 )

Cloning of the orf6 and orf7 genes, and preparation 660 1 of recombinant ORF6 and ORF7 The orf 6 and -7 genes were amplified by PCR and 0.1 cloned to pET, and recombinant ORF -6 and -7 proteins AguA(ORF6) AguB(ORF7) were expressed as C-terminal His -tag fusion proteins in Growth (OD 6 0.01 E. coli Rosetta (DE3). Recombinant proteins were 0215791134 6 81012 purified by Ni2þ chelating affinity, DEAE-5PW, and phenyl-5PW column chromatographies to electropho- Time (h) retic homogeneity. The enzyme activities of the purified Fig. 6. Western Blotting Analysis of Crude Extract from S. rumi- recombinant ORF6 and ORF7 preparations were exam- nantium for the Presence of AguA and AguB. ined using agmatine and NCP respectively as substrates S. ruminantium cells were grown in TYG medium. At the times (Fig. 5). Agmatine was converted to a ninhydrin-posi- indicated in this figure, cells were harvested and disrupted by tive , showing migration similar to that of sonication. The sonicated extract was analyzed by SDS–PAGE, followed by Western blotting for the presence of AguA and AguB, authentic NCP, by rORF6 (Fig. 5). The reaction product using anti-rAguA and -rAguB antisa respectively. from agmatine by rORF6 was analyzed with an API 2000 mass spectrophotometer. Its molecular mass was 131.8 Da identical to that of authentic NCP. NCP was indicating that both aguA and aguB were expressed in converted to putrescine by ORF7 (Fig. 5). From these S. ruminantium. results, ORFs 6 and -7 were confirmed as agmatine deiminase and NCP amidohydrolase respectively. Properties of rAguA and rAguB Hence, orfs6 and -7 were designated the agmatine The molecular masses of S. ruminantium AguA and deiminase gene (aguA) and the NCP amidohydrolase AguB were calculated to be 41,391 Da and 32,532 Da gene (aguB), respectively. respectively from their predicted amino acid sequences. The molecular mass of the rAguA preparation as Expression of aguA and aguB in S. ruminantium measured by SDS–PAGE and gel filtration analysis Expression of aguA and aguB in S. ruminantium cell was 45 kDa. On the other hand, the determined molecu- was examined by Western blotting analysis using anti- lar mass of the rAguB preparation was 37 kDa by SDS– rAguA and -rAguB antisera (Fig. 6). Both AguA and PAGE and the 80 kDa by gel filtration analysis. The data AguB were detected in the cells grown in TYG medium, show that S. ruminantium rAguA and rAguB consisted 454 S. LIAO et al. of a monomer and a homodimer respectively. AguAs Acknowledgments from Pseudomonas aeruginosa,31) Arabidopsis thali- ana,32) shoots,33) and rice34) have been reported to This work was supported in part by the Foundation of be homodimers consisting of 43, 43, 44, and 95 kDa the Noda Institute for Scientific Research. S. Liao and subunits respectiverly, except that from soy bean, with a K-C. Ko were supported by pre-doctoral fellowship monomer of 70 kDa.35) AguBs from P. aeruginosa and from Yoneyama Foundation and Waroh Suzuki Foun- A. thaliana have been reported to consist of homohex- dation respectively. P. Poonpairoi was the recipient of a amers consisting of subunits of 33 kDa, and 35 kDa fellowship from the Ministry of Education, Cultures, respectively.3,31,36) Sports, Science and Technology of Japan. Y. Takatsuka The optimum pH and temperature for rAguA were 6.5 and Y. Yamaguchi were the recipients of postdoctoral and 45 C, respectively. The enzyme was stable for fellowships from the Japan Society for Promotion of 30 min in a pH range of 6.0 to 9.0 and at temperatures Science. below 40 C. Optimum pH for rAguB activity was about 7.0, and the enzyme was stable from pH 6.0 to 8.0. The optimum temperature for rAguB was 45 C. AguB References activity was stable at up to 70 C for 30 min in 50 mM PIPES buffer (pH 6.5). 1) Hirao, T., Sato, M., Shirahata, A., and Kamio, Y., The Km and Vmax values obtained from a Line- Covalent linkage of polyamines to peptidoglycan in weaver-Burk plot were 6.67 mM and 1.22 nmol/min/mg Anaerovibrio lypolytica. J. Bacteriol., 182, 1154–1157 protein of rAguA towards agmatine and 0.22 mM and (2000). 0.33 nmol/min/mg protein of rAguB towards NCP 2) Kamio, Y., Itoh, Y., Terawaki, Y., and Kusano, T., respectively. AguA from rice seedlings showed high Cadaverine is covalently linked to peptidoglycan in Km (15 mM),4) while the Km values for AguA from Selenomonas ruminantium. J. Bacteriol., 145, 122–128 P. aeruginosa27) A. thaliana,3) maize shoots,33) soy- (1981). 35) 37) 3) Kamio, Y., and Nakamura, K., Putrescine and cadaver- bean, and cucumber were lower than 1 mM. 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