Abstract
The leucine dehydrogenase (L-leucine: NAD+ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli-pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350, 000 and consists of six subunits identical in molecular weight (56, 000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15min; at 55°C and various pH's between 6.0 and 10.0 for 10min. The enzyme catalyzes the oxidative deamination of branched-chain L-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD+ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].
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