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Journal of Bacteriology, August 2003, p. 4483-4489, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4483-4489.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Novel Psychrophilic and Thermolabile L-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1

Takayuki Kazuoka,¹ Shouhei Takigawa,¹ Noriaki Arakawa,¹ Yoshiyuki Hizukuri,¹ Ikuo Muraoka,¹ Tadao Oikawa,^1,2* and Kenji Soda¹

Department of Biotechnology, Faculty of Engineering, Kansai University,¹ Kansai University High Technology Research Center, Suita, Osaka-fu 564-8680, Japan²

Received 12 September 2002/ Accepted 17 April 2003

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD⁺-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD⁺ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K_m for L-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD⁺ binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.

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* Corresponding author. Mailing address: Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka-fu 564-8680, Japan. Phone: 81-6-6368-0812. Fax: 81-6-6388-8609. E-mail: oikawa{at}ipcku.kansai-u.ac.jp.

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Journal of Bacteriology, August 2003, p. 4483-4489, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4483-4489.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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