Kamegaya Taichi, Kuroda Kenji, Hayakawa Yoshihiro
Graduate School of Medicine, Nagoya University.
Nagoya J Med Sci. 2011 Feb;73(1-2):49-57.
Here we show that bis(3'-5') cyclic diadenylic acid (c-di-AMP) and a diadenylate cyclase (DAC) domain protein involved in the biosynthesis of c-di-AMP were identified in Streptococcus pyogenes. The matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrum of the cell extract of S. pyogenes, which showed a fragment pattern very similar to that of the authentic sample of c-di-AMP, revealed that S. pyogenes produces c-di-AMP in the cell. Subsequently, we confirmed by an in vitro experiment that the production of c-di-AMP in the cell is due to the action of Spy1036 gene encoding a DAC domain protein named spyDAC, which is a new protein different from a well-known diadenylate cyclase. Moreover, the experiment gave a product with a molecular weight of 657.021, which is consistent with the molecular weight of c-di-AMP. Furthermore, the mass spectral fragment pattern of the product obtained by the in vitro biosynthesis is quite similar to that of the product produced by the above in vivo experiment. This in vitro production of c-di-AMP indicated that spyDAC in S. pyogenes actually catalyzes the in vivo biosynthesis of c-di-AMP from ATP.
在此我们表明，在化脓性链球菌中鉴定出了双（3'-5'）环二腺苷酸（c-di-AMP）以及参与c-di-AMP生物合成的二腺苷酸环化酶（DAC）结构域蛋白。化脓性链球菌细胞提取物的基质辅助激光解吸电离（MALDI）-飞行时间（TOF）质谱显示出与c-di-AMP真实样品非常相似的片段模式，这表明化脓性链球菌在细胞内产生c-di-AMP。随后，我们通过体外实验证实，细胞内c-di-AMP的产生是由于编码名为spyDAC的DAC结构域蛋白的Spy1036基因的作用，该蛋白是一种不同于已知二腺苷酸环化酶的新蛋白。此外，该实验得到了分子量为657.021的产物，这与c-di-AMP的分子量一致。此外，通过体外生物合成获得的产物的质谱片段模式与上述体内实验产生的产物非常相似。c-di-AMP的这种体外产生表明，化脓性链球菌中的spyDAC实际上催化了由ATP进行的c-di-AMP体内生物合成。
