Functional characterization of Zymomonas mobilis exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GppA) protein

Abstract

Inorganic polyphosphate (poly-P) molecules are linear polymers of orthophosphate units linked by ‘high-energy’ phosphoanhydride bonds. In bacteria, poly-P is involved in many physiological processes such as cellular metabolism, growth, motility, ‘stress-responses’, virulence, quorum sensing and biofilm formation. Most bacterial species encode members of the exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) protein family to hydrolyze intracellular poly-P. However, PPX/GPPA proteins exhibit considerable heterogeneities in their amino acid (aa) sequence compositions and biochemical capabilities. Here, we report the detailed biochemical characterization of the 508 aa PPX/GPPA homologue encoded within the ethanol-producing bacterium Zymomonas mobilis subsp. mobilis NCIMB 11163 (ZmPPX; Za10_0559). Our results reveal that ZmPPX possesses efficient exopolyphosphatase, GTPase, ATPase and pppGpp 5’-phosphohydrolase activities (i.e. conversion of pppGpp to ppGpp). Unlike most other PPX/GPPA homologues, ZmPPX does not exhibit poly-P chain length specificity, as indicated by its respective enzymatic kinetic parameters (Vmax, Km and kcat). Regarding its nucleotide hydrolyzing activities, ZmPPX prefers GTP over ATP. Inhibition studies showed that both ppGpp and pppGpp alarmones effectively inhibited the exopolyphosphatase activities of ZmPPX; with 200 micromolar concentrations of ppGpp and pppGpp reducing the rate of poly-P hydrolysis by ca. 90% and ca. 70%, respectively. Increasing the concentration of poly-P substrate did not relieve the inhibition by ppGpp or pppGpp, suggesting that the alarmones were non-competitive inhibitors of ZmPPX exopolyphosphatase activities. Inhibition constants (Ki) of both alamones were determined and their Ki values indicated that ppGpp was a stronger inhibitor of ZmPPX exopolyphosphatase activity than pppGpp. This is contrary to the situation observed for other PPX/GPPA proteins studied to date, where pppGpp is the more potent inhibitor. In conclusion, our results indicate that ZmPPX most likely functions as the major protein responsible for polyphosphate hydrolysis in Z. mobilis. Our biochemical data also suggests that the stringent response may operate, and be modulated via, subtly different molecular mechanisms in Z. mobilis, compared to other bacterial species studied to date. This notable finding requires further detailed biological investigation. Funding statement: This work was supported by the Research Grants Council (RGC) of Hong Kong, via a GRF grant (# 17121814) to R.M.W.