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.Various psychrophilic and psychrotrophic microorganisms widely occur in natural and artificial environments, such as in cold rooms and refrigerated transport systems. They take part in the natural turnover of a variety of organic and inorganic compounds under cold conditions (13). In addition to L-threonine dehydratase and L-threonine aldolase, which are pyridoxal enzymes, L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) plays an important role in L-threonine catabolism.L-ThrDH catalyzes the NAD-dependent dehydrogenation of L-threonine to L-2-amino-3-oxobutyrate, which spontaneously decomposes to aminoacetone and CO 2 (15) or is cleaved thiolytically by 2-amino-3-oxobutyrate coenzyme A lyase to glycine and acetyl coenzyme A (14). D-ThrDH exclusively catalyzes an analogous reaction with D-threonine (16). The dehydrogenation catalyzed by L-ThrDH occurs at the ␤-position of L-threonine, although other amino acid dehydrogenases (3,8,(18)(19)(20) catalyze the ␣-deamination reactions. L-ThrDH is regarded as a kind of alcohol dehydrogenase in ...