This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ono, H. Articles by Murooka, Y. Search for Related Content PubMed PubMed Citation Articles by Ono, H. Articles by Murooka, Y.

Journal of Bacteriology, January 1999, p. 91-99, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

Hisayo Ono,^* Kazuhisa Sawada, Nonpanga Khunajakr, Tao Tao, Mihoko Yamamoto, Masayuki Hiramoto, Atsuhiko Shinmyo,^§ Mitsuo Takano, and Yoshikatsu Murooka

Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita-shi, Osaka 565-0871, Japan

Received 27 April 1998/Accepted 13 October 1998

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic -semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had K_ms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to -N-acetyl-,-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a K_m of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

---

^* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita-shi, Osaka 565-0871, Japan. Phone: 81 6 877 5111, ext. 3432. Fax: 81 6 879 7418. E-mail: ono{at}res.bio.eng.osaka-u.ac.jp.

Present address: Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand.

Present address: Department of Biology, Wuhan University, Hubei 410072, People's Republic of China.

^§ Present address: Department of Biotechnology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-01, Japan.

---

Journal of Bacteriology, January 1999, p. 91-99, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Bursy, J., Pierik, A. J., Pica, N., Bremer, E. (2007). Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase. J. Biol. Chem. 282: 31147-31155 [Abstract] [Full Text]  
• Schubert, T., Maskow, T., Benndorf, D., Harms, H., Breuer, U. (2007). Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium. Appl. Environ. Microbiol. 73: 3343-3347 [Abstract] [Full Text]  
• Jebbar, M., Sohn-Bosser, L., Bremer, E., Bernard, T., Blanco, C. (2005). Ectoine-Induced Proteins in Sinorhizobium meliloti Include an Ectoine ABC-Type Transporter Involved in Osmoprotection and Ectoine Catabolism. J. Bacteriol. 187: 1293-1304 [Abstract] [Full Text]  
• Vandenende, C. S., Vlasschaert, M., Seah, S. Y. K. (2004). Functional Characterization of an Aminotransferase Required for Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa PAO1. J. Bacteriol. 186: 5596-5602 [Abstract] [Full Text]  
• Calderon, M. I., Vargas, C., Rojo, F., Iglesias-Guerra, F., Csonka, L. N., Ventosa, A., Nieto, J. J. (2004). Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T. Microbiology 150: 3051-3063 [Abstract] [Full Text]  
• Pflughoeft, K. J., Kierek, K., Watnick, P. I. (2003). Role of Ectoine in Vibrio cholerae Osmoadaptation. Appl. Environ. Microbiol. 69: 5919-5927 [Abstract] [Full Text]  
• Kuhlmann, A. U., Bremer, E. (2002). Osmotically Regulated Synthesis of the Compatible Solute Ectoine in Bacillus pasteurii and Related Bacillus spp.. Appl. Environ. Microbiol. 68: 772-783 [Abstract] [Full Text]  
• Nakayama, H., Yoshida, K., Ono, H., Murooka, Y., Shinmyo, A. (2000). Ectoine, the Compatible Solute of Halomonas elongata, Confers Hyperosmotic Tolerance in Cultured Tobacco Cells. Plant Physiol. 122: 1239-1248 [Abstract] [Full Text]  
• Cánovas, D., Borges, N., Vargas, C., Ventosa, A., Nieto, J. J., Santos, H. (1999). Role of Ngamma -Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine. Appl. Environ. Microbiol. 65: 3774-3779 [Abstract] [Full Text]