polyphosphate substrates. Its activities appear to be similar to those of the CG-PPX2 protein from C. glutamicum. Both enzymes prefer short-chain polyphosphate substrates, require Mg2+ or Mn2+ ion order WP 1130 cofactors, and have optimal activities at slightly acidic pH values. The sequences of the MTB-PPX1, Rv1026 and CG-PPX2 proteins align with the N-terminal regions of the E. coli PPX and GPP Biochemical Activities of Rv0496 and Rv1026 proteins. The available structural and bioinformatic data indicates that polyphosphate and pppGpp substrates are hydrolyzed at a single active site located within this region. Both the MTB-PPX1 and Rv1026 proteins contain conserved amino acid residues implicated in the catalytic mechanism. Asp135 and Glu142 and Asp146 and Glu153 are predicted to be responsible for binding the essentially-required Mn2+ or Mg2+ ions. Arg84 and Glu112 and Arg90 and Glu123 are implicated in the hydrolysis of the phosphoanhydride bond linking the terminal and penultimate phosphate units in the poly-P or ATP substrates. X-ray crystal structures of the E. coli PPX protein suggest that the polyphosphate chain is bound primarily by residues located within the C-terminal region. This is supported by biochemical and biophysical data obtained from truncated forms of the EC-PPX protein lacking the C-terminal domain. As these ca. 150200 C-terminal residues are absent in MTB-PPX1, it most likely adopts an alternative mode of polyphosphate binding. This may be responsible for the differences in polyphosphate chain length preferences observed for MTB-PPX1, compared to the E. coli PPX and GPP proteins; which have higher affinities for short and longchain polyphosphate substrates, respectively. Bolesch and Keasling 
previously defined the N-terminal ca. 300 residues of EC-PPX as functioning as a `quasi-processive’ exopolyphosphatase. This is what the MTB-PPX1 protein appears to function as. The activities of additional diverse PPX-GppA homologues will need to be characterized to determine whether this putative structure-function relationship generally holds true. Our finding that the E. coli GPP protein has notable GTPase activities is not consistent with the previous report by Hara and Sy. We tentatively speculate that these authors may have inadvertently purified and characterized the E. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22203538 coli PPX protein; as their report predates the identification and characterization of ECPPX; and the discovery that this protein also possesses guanosine pentaphosphate 59-phosphohydrolase activities. Future investigations may substantiate or repudiate this speculation. Transposon mutagenesis has previously shown that the rv0496 gene was non-essential in the H37Rv strain, whilst rv1026 was required for optimal growth. Rv0496 was found to be secreted from M. tuberculosis cells, and was identified as a T-cell antigen with potential for vaccine development. Thayil et al. have recently reported that the 344 aa MT0516 protein from M. tuberculosis CDC1551, which has an identical sequence to Rv0496 from the H37Rv strain, could hydrolyze a 65-mer of polyphosphate; noting that its polyphosphate hydrolase activities were not inhibited in the presence of 1 mM of ppGpp. The authors also reported that the intracellular poly-P levels were elevated in a MT0516-deficient strain of M. tuberculosis during the mid-logarithmic and late stationary growth phases. Using a guinea pig lung model, they further demonstrated that the activities of MT0516 played a key role in enabling M. tuberc
