Nessi C, Albertini A M, Speranza M L, Galizzi A
Dipartimenti di Genetica e Microbiologia, Università di Pavia, Italy.
J Biol Chem. 1995 Mar 17;270(11):6181-5. doi: 10.1074/jbc.270.11.6181.
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 NH3-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 NH3-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 degrees 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 NH4+. 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.
枯草芽孢杆菌的outB基因参与孢子萌发和生长，对生长至关重要。通过在大肠杆菌中表达获得了OutB蛋白，并纯化至表观均一。在此我们报告实验表明，OutB是一种依赖氨的NAD合成酶，该酶催化NAD生物合成的最后一步反应。该酶由两个相同的30240 Da亚基组成，依赖氨，而谷氨酰胺作为酰胺供体效率较低。NAD合成酶对热高度耐受，在100℃下失活半衰期为13分钟。从一株在孢子萌发和生长过程中对温度敏感的枯草芽孢杆菌菌株中纯化出一种突变型NAD合成酶。该突变酶的活性比野生型低200倍，最适温度较低，对铵离子的动力学曲线不是双曲线。OutB的合成时间进程表明，该酶的合成在萌发和生长过程中开始，并在指数生长末期达到最高水平。该酶可从休眠孢子中回收。
