Versatile enzymatic system for the production of guanosine polyphosphates

Abstract

During periods of environmental stress, bacteria synthesize guanosine tetraphosphate (ppGpp, magic spot I) and guanosine pentaphosphate (pppGpp, magic spot II) in a process known as the stringent response. These intracellular allarmone molecules ‘reprogramme’ the transcriptional and translational machinery to help the cell conserve scarce resources. Existing methods for the production of guanosine polyphosphates are either technically difficult or inefficient, hindering investigations into their biological roles. We have developed a simple and efficient one-step enzymatic method for the production of guanosine polyphosphates using a recombinant protein cloned from Staphylococcus aureus. The purified enzyme efficiently catalyses the formation of pppGpp (and AMP) from GTP + ATP; and ppGpp (and AMP) from GDP + ATP. Notably, it also catalyses the synthesis of pGpp (guanosine 5’-monophosphate 3’-diphosphate, and AMP) from GMP + ATP; albeit with reduced efficiency. The reverse reactions are not catalysed, leading to high conversion rates. Guanosine polyphosphate products can be obtained in a homogeneous form using a combination of anion exchange chromatography followed by desalting. Our approach can be used to produce guanosine polyphosphates on a multi-milligram scale. Furthermore, our results also suggest that a third ‘magic spot’ allarmone may be formed within certain bacterial species.