EAPSI:Investigating Roles of Putative Phosphonatases from the Extreme Bacterium Synechococcus

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Phosphorus is the eleventh most abundant element on the planet, and essential to life. Phosphate esters, compounds that contain a C-O-P bond, make up the backbones of DNA and RNA and are also a component of the molecular energy molecule ATP. Thus, phosphate esters have been well studied for many decades. Less thoroughly examined are organophosphanates, compounds which contain a C-P bond. Organophosphonates are thought to have predominated on primitive Earth, when oxygen concentration was likely lower than today. More recently, organophosphonates have been noted for their importance as commercial products, from antibiotics to fungicides, and in providing nutrients for organisms living in extreme environments. Additionally, organophosphonates have recently been recognized as an important component of the marine phosphorus cycle, accounting for up to 25% of oceanic dissolved organic phosphate. Understanding organophosphonate breakdown and utilization is thus important for understanding environmental phosphorus cycling. Work will be conducted with Professor Taichi Takasuka, at Hokkaido University, to make and test the function of enzymes proposed to be able to break down these compounds. It is possible that they have the ability to break down organophosphonates in a way that has never been reported before. The most well known microbial pathway for organophosphonate catabolism is that of C-P lyase, a multi-subunit enzyme complex that, in all bacteria studied, is under control of a single, phosphate-activated promoter. C-P lyase?s mechanism has recently been proposed, along with mechanisms for alternative organophosphonate catabolism. Of these alternative pathways, most have been at least partially characterized. However, three recent publications present evidence for as-yet-uncharacterized catabolic pathways of phosphonate degradation in Campylobacter spp., Heliobacter spp., and Synechococcus spp. Specifically, in Synechoccus spp. isolated from Yellowstone National Park, gene products have been implicated in organophosphonate catabolism, but neither verified nor characterized. These putative phosphonatases will be expressed using a wheat germ embryo-based cell-free protein production system, and biochemically characterized, using quantitative mass spectrometry (MS). The putative phosphonatases (phnases) implicated in phosphonate catabolism by this study and their metabolic products may reveal novel mechanisms of breakdown of this important subset of phosphate containing compounds. This NSF EAPSI award is funded in collaboration with the Japan Society for the Promotion of Science.