Objectives: To elucidate the functional roles of Rv0496 and Rv1026 in the metabolism of polyphosphate and guanosine polyphosphate molecules.
Methods: Rv0496 and Rv1026 were expressed as N-terminal MalE-fusions in E. coli BL21(DE3), and purified by an efficient 2-step procedure. The proteins were analyzed using a range of biophysical and biochemical methods to establish their exopolyphosphatase, pppGpp hydrolase and nucleotide polyphosphate hydrolase activities.
Results: Rv0496 functioned as an exopolyphosphatase, but Rv1026 had no poly-P-related activities. Neither enzyme possessed pppGpp hydrolase activities. Furthermore, unlike the E. coli Ppx enzyme, ppGpp did not modulate exopolyphosphatase activities. Intriguingly, both proteins had various nucleotide triphosphate hydrolase activities.
Conclusions: The M. tuberculosis Rv0496 protein should be placed within the Ppx family, not the Gppa-ppx family. Rv1026 is neither a Ppx nor a Gppa-Ppx protein, and its NTPase activities indicate it may have other metabolic functions within M. tuberculosis cells.
Funding: Research Grants Council of Hong Kong, GRF grant (#705007) to RMW