Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS-3

Leucine dehydrogenase ( -leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) was purified to homogeneity from the crude extract of an alkaliphilic halophile, Natronobacterium magadii MS-3, with a yield of 16%. The enzyme had a molecular mass of about 330 kDa and consisted of six subunits identical in molecular mass (55 kDa). The enzyme required a high concentration of salt for stability and activity. It retained the full activity after heating at 50 °C for 1 h and about 50% activity after being kept at 30 °C for 2 months in the presence of 2.5 M NaCl. The enzyme required NAD⁺ as a coenzyme and showed maximum activity in the presence of more than 3 M salt, as CsCl, RbCl, NaCl, or KCl. In addition to -leucine, -valine and -isoleucine were also good substrates in the oxidative deamination. In the reductive amination, 2-keto analogs of branched-chain amino acids were substrates. The Michaelis constants were 0.69 mM for -leucine, 0.48 mM for NAD⁺, 4.0 mM for 2-ketoisocaproate, 220 mM for ammonia, and 0.02 mM for NADH in the presence of 4 M NaCl. The K_m for -leucine depended on the concentration of salt and increased with decreasing salt concentration. The N. magadii enzyme was unique in its halophilicity among leucine dehydrogenases studied so far.