Purification and Characterization of Alanine Dehydrogenase from a Cyanobacterium, Phormidium lapideum

Abstract

Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N₂-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH₂ terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD⁺, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The K_m, values were 5.0mM for L-alanine and 0.04mM for NAD⁺, 0.33mM for pyruvate, 60.6mM for NH₄⁺ (pH 8.7), and 0.02mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP.