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Journal of Bacteriology, February 2008, p. 1359-1365, Vol. 190, No. 4
0021-9193/08/$08.00+0 doi:10.1128/JB.01184-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon, Pyrobaculum islandicum
,
Masato Ohnishi,1
Makoto Saito,1
Sadao Wakabayashi,2
Morio Ishizuka,3
Katsushi Nishimura,4
Yoko Nagata,1* and
Sabu Kasai5*
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo 101-8308, Japan,1 Department of Life Sciences, Graduate School of Life Science, University of Hyogo, Kamigohri, Hyogo, Japan,2 Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, Bunkyo-Ku, Tokyo, Japan,3 Department of Applied Chemistry, Junior College, Nihon University, Funabashi, Chiba, Japan,4 Department of Applied Chemistry and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto, Sumiyoshi, Osaka 558-8585, Japan5
Received 26 July 2007/ Accepted 12 October 2007
Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100°C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95°C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 µmol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 µmol/min/mg, respectively.
* Corresponding author. Mailing address for Y. Nagata: Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-14 Kanda-Surugadai, Chiyoda Ward, Tokyo 101-8308, Japan. Phone: 81-3-3259-0861. Fax: 81-3-3293-7572. E-mail: nagata{at}chem.cst.nihon-u.ac.jp. Mailing address for S. Kasai (DNA cloning and sequencing questions): Department of Applied Chemistry and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto, Sumiyoshi, Osaka 558-8585 Japan. Phone and Fax: 81-6-6605-2783. E-mail: kasai{at}bioa.eng.osaka-cu.ac.jp
Published ahead of print on 26 October 2007.
Supplemental material for this article may be found at http://jb.asm.org/.
Journal of Bacteriology, February 2008, p. 1359-1365, Vol. 190, No. 4
0021-9193/08/$08.00+0 doi:10.1128/JB.01184-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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