High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

Abstract

BackgroundWe recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities.Methodology/Principal FindingsRecombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility.Conclusions and General SignificanceWe show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling.

Crystal structure and biochemical analyses reveal that the Arabidopsis triphosphate tunnel metalloenzyme AtTTM3 is a tripolyphosphatase involved in root development

“…The addition of inorganic phosphatase led to a further increase in P i formation, confirming that AtTTM3 hydrolyzed PPP i into P i and PP i and did not further hydrolyze PP i (data not shown). Similarly to other TTM proteins, 2.5 m m Ca 2+ inhibited Mg 2+ ‐dependent PPPase activity (Figure b; Delvaux et al ., ; Kohn et al ., ).…”

“…Furthermore, Kohn et al . () detected trace amounts of PPP i in E. coli , concluding that the concentration must be in the low micromolar or nanomolar range. Thus, the lack of evidence for the presence of PPP i in cells might simply be an issue of detection sensitivity.…”

“…While CthTTM was also able to hydrolyze a longer polyphosphate substrate (Jain and Shuman, ), AtTTM3, like NeuTTM and E. coli ygiF, did not display significant activity towards a long‐chain polyphosphate (Figure ; Delvaux et al ., ; Kohn et al ., ), raising the question of what the physiological function of AtTTM3 or, generally, TTMs with PPPase activity is. The existence of PPP i in living cells is still unclear.…”

Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer

“…When nucleoside triphosphohydrolase activity is thermodynamically linked with hydrolysis of PPP i to 3P i by cellular triphophosphatases, the overall process is exergonic and can only be reversed with great energetic expense by the cell 29 . Thus, it would be expected that the activity of SAMHD1 would be regulated at multiple levels.…”

GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state

“…The tripolyphosphate is likely degraded to 3P i through the action of plentiful cellular pyrophosphatases or tripolyphosphatases (14). Presumably, degradation to the level of nucleoside rather than deoxynucleoside diphosphate (dNDP) or deoxynucleoside monophosphate (dNMP) is to make the process energetically or kinetically costly to reverse, with the additional possibility that the neutral nucleoside will be irreversibly transported out of the cell (9).…”