Purification, properties, and metabolic roles of NAD⁺-glutamate dehydrogenase in Clostridium botulinum 113B

Abstract

Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD⁺-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of >1,092 μmolxmin^-1xmg protein^-1. The enzyme is a hexamer of a polypeptide of Mr=42,500, and the native molecular weight is 250,800. The apparent K _m values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD⁺ in the deamination reaction, and 7.2 mM for α-ketoglutarate, 243 mM for NH ⁺₄ and 0.028 mM for NADH in the reverse reaction. NADP⁺ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate α-ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.