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Occurrence of Agmatine Pathway for Putrescine Synthesis in Selenomonas Ruminatium

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Occurrence of Agmatine Pathway for Putrescine Synthesis in Selenomonas Ruminatium Biosci. Biotechnol. Biochem., 72 (2), 445–455, 2008 Occurrence of Agmatine Pathway for Putrescine 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 Nutrition, 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 cadaverine and Polyamines such as putrescine, cadaverine, and putrescine from L-lysine and L-ornithine as the essential spermidine are essential constituents of peptidoglycan constituents of its peptidoglycan by a constitutive lysine/ and they play a significant role in the maintenance of the ornithine decarboxylase (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 arginine 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 enzyme in agmatine pathway for cadaverine respectively link covalently to the -carbox- putrescine synthesis, in S. ruminantium. We purified yl group of the D-glutamic acid residue of the peptido- and characterized ADC and cloned its gene (adc) from glycan, which is catalyzed by diamine:lipid intermediate S. ruminantium chromosomal DNA. ADC showed more transferase, 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 bacteria, 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, encoding agmatine deiminase boxylase activities towards both L-lysine and L-ornithine 8) (AguA) and N-carbamoyl-putrescine amidohydrolase 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 Food 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 Biochemistry, 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 spermine 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 protein 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- proteolysis 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-yeast extract (1%)-glucose (0.5%) medium sup- binding to the P22-binding site 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 amino acid 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 Â 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 decarboxylation 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 enzymes, T4 DNA ligase, 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.
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