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

Myxococcus xanthus generates diadenosine tetraphosphates (ApA) and diadenosine pentaphosphates (ApA) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes ApA from ATP, ApA from ATP and adenosine tetraphosphate (Ap), and Ap from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze ApA and Ap 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 ApA or Ap synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest ApA 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 Ap from ATP and triphosphate in the absence of pyrophosphatase. Ap formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of ApA, 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 Ap from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap from ATP and 3-phosphoglycerate in the presence of Mg and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap synthesis in M. xanthus.