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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,dagger Tao Tao,Dagger 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 beta -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 Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma -N-acetyl-alpha ,gamma -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 Km 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.

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

Dagger 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.


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