Purification and characterization of leucine dehydrogenase from the thermophile 'Bacillus caldolyticus'

Abstract

The L-leucine dehydrogenase (LeuDH, EC 1.4.1.9) from the extremely thermophilic 'Bacillus caldolyticus' (DSM 405) has been purified to homogeneity and crystallized. Its physical and biochemical properties, such as subunit structure, M(r), amino acid composition, isoelectric point, heat stability, pH-optima, K(m) values for coenzymes and substrates, and pattern of inhibition have been determined. The native enzyme is an octamer (M(r) 320 000) composed of identical subunits (M(r) 39 000) which contain one intrachain disulphide bridge. Only aliphatic amino acids were accepted as substrates and a preference was exhibited for branched-chain residues. Inhibition occurred only upon incubation with thiol reagents such as HgCl2 and p-mercuribenzoate, and pyridoxal 5'-phosphate. The LeuDH was very thermostable, with 50% residual activity remaining after 30 min incubation at 80°C. Its properties, as well as amino acid composition and N-terminal sequence, were compared with those of the phylogenetically related LeuDH from the mesophile Bacillus cereus. Both enzymes displayed very similar properties: the quaternary structures were identical, amino acid composition alike, the N-terminal sequence showed 62% homology for the first 19 residues, and K(m) values for the different substrates differed by a factor of two or less, indicating that the active centres had a similar structure. Thus, the only major difference observed was the much higher stability of the thermophilic enzyme to denaturation by heat and organic solvents.