Dihydrodipicolinic acid synthase of Bacillus licheniformis. Quaternary structure, kinetics, and stability in the presence of sodium chloride and substrates.

Dihydrodipicolinic acid synthase (L-aspartate-beta-semialdehyde hydro-lyase (adding pyruvate and cyclising), EC 4.2.1.52) obtained from Bacillus licheniformis was purified to homogeneity. Its molecular weight was 108 000 to 117 500, depending on the concentration of NaCl and substrates present, and it contained four subunits of identical molecular weight (28000). The Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 and 2.6 mM, respectively. It was previously shown that pyruvate and a high sodium chloride concentration contributed to the stability of the enzyme. The effect of these substances and the other substrate, L-aspartic semialdehyde, on molecular weight was determined. None of these three substances significantly affected the apparent molecular weight. The effect of sodium chloride, pyruvate, and L-aspartic semialdehyde on enzyme structure was studied by determining the effect of their presence on inactivation of the enzyme by several chemical denaturants and heat. Pyruvate dramatically protected against inactivation by all of the denaturants. Sodium chloride protected against inactivation by sodium dodecyl sulfate, guanidine-HCl, urea, and heat, but somewhat facilitated inactivation by ethanol. L-Aspartic semialdehyde had no significant effect on inactivation by sodium dodecyl sulfate and ethanol; it rendered the enzyme slightly more sensitive to inactivation by guanidine-HCl and urea. The thermal melting curve obtained for the enzyme in the presence of L-aspartic semialdehyde was biphasic. The activity was reduced approximately 50% by heating for 30 min at temperatures between 50 and 80 degrees C. Only by heating at temperatures above 80 degrees C did the inactivation become complete. The partially inactivated enzyme could be reactivated by heating after removal of the L-aspartic semialdehyde. Pyruvate prevented the partial inactivation and facilitated reactivation. The only difference detected between the native enzyme and the partially inactivated form of the enzyme was that the latter had a reduced V. It is known that in other spore-formers, dihydrodipicolinate synthase increases in activity late in sporulation. This increase may be important for normal sporulation to occur. The possibility is discussed that the intracellular pool sizes of pyruvate and L-aspartic semialdehyde might have an influence on the level of dihydrodipicolinate synthase activity, by controlling the amount of partial inactivation of the enzyme that occurs in vivo.