Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus.

Myxococcus xanthus generates diadenosine tetraphosphates (Ap4A) and diadenosine pentaphosphates (Ap5A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap4A from ATP, Ap5A from ATP and adenosine tetraphosphate (Ap4), and Ap4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap4A and Ap4 synthesis, 15 M. xanthus aminoacyl-tRNA synthetases (aaRSs), four acyl-CoA synthetases (Acys), three acetyl-CoA synthetases (Aces), phosphoglycerate kinase (Pgk), and adenylate kinase (Adk) were expressed in Escherichia coli and examined for Ap4A or Ap4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap4A synthetase activity. AlaRS, SerRS, and LeuRS1 showed high ADP synthetase activity with ATP as a substrate in the presence of pyrophosphatase, and also demonstrated the ability to produce Ap4 from ATP and triphosphate in the absence of pyrophosphatase. Ap4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap4A, suggesting that these enzymes prefer triphosphate over ATP as a substrate in the second reaction. Some of the recombinant M. xanthus Acys and Aces also synthesized Ap4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap4 from ATP and 3-phosphoglycerate in the presence of Mg2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap4 synthesis in M. xanthus.