The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth. The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity. Here we report experiments showing that OutB is a NH-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD. The enzyme is composed of two identical subunits of 30,240 Da and is NH-dependent, whereas glutamine is inefficient as an amide donor. The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 °C of 13 min. A mutant NAD synthetase was purified from a B. subtilis strain temperature-sensitive during spore germination and outgrowth. The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH. The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth. The enzyme could be recovered from dormant spores.

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