MsPrsA activation by Pi and Mg²⁺ ions.

<p>The enzyme activity was measured based on the NADH-coupled enzymatic assay described in Materials and Methods. (A) The MsPrsA activity was measured at pH 8.0 Tris HCl 50mM, varying the concentration of potassium phosphate at pH 7.5, between 1 to 150mM. (B) The enzyme activity was measured at pH 8.0 Tris HCl 50mM, varying the magnesium concentration from 0.5 to 20mM, at 5 mM R5P and 0.5 mM Mg-ATP fixed concentrations. Results are the mean values of three replicates.</p

Structural comparison of human and <i>M</i>. <i>smegmatis</i> PrsA.

<p>(A) Superimposed structures with hPrsA-I (PDB: 2HCR) shown in light blue and MsPrsA with a white-red colour and sausage representation scheme according to the degree of amino-acids sequence conservation defined as follows: red, conserved; white, non-conserved; sausage radius proportional to the sequence alignment score: the smaller the radius the higher the conservation. Close-up views of the R5P binding site (B), the flexible loop part of the ATP binding site (C) and the allosteric regulatory site (D). Same color scheme as above and sausage representation omitted for clarity.</p

Sequence alignment of PrsA from <i>Mycobacterium smegmatis</i> (MsPrsA), <i>Mycobacterium tuberculosis</i> H37Rv (MtPrsA) <i>Bacillus subtilis</i> (BsPrsA), and human isoform 1 (hPrsA-1).

<p>Conserved residues are shown on a red background whereas chemically similar residues on a yellow background. The sequence identities between MsPrsA, MtPrsA, BsPrsA, and hPrsA are 88%, 46%, 43% respectively. Numbering and secondary structure elements assignments are shown for the mycobacterial enzyme. Consensus sequence information are edited according to the Espript3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175815#pone.0175815.ref019" target="_blank">19</a>] notation conservation.</p

Primer sequences (5’-3’) used in this study.

<p>Primer sequences (5’-3’) used in this study.</p

The overall structure of MsPrsA.

<p>Ribbon representation of the quaternary structure consisting of an hexameric arrangement (A) and of the monomer fold (B). Domains are colored in cyan (N-terminal) and green (C-terminal). The 3₁₀-helix and the flexible loop, part of the N-terminal domain, are colored in red and magenta, respectively. Flag regions (β-hairpin β2-β3, and β9-β10) are shown in yellow, and the two extremities of the disordered loop are indicated (residues 204 and 213). The acetate molecule (ACT) is shown as ball and stick and the water is represented as a red sphere. (C) Magnified view of R5P binding loop with protein residues and acetate shown as ball-and-stick and the water molecule as a red sphere; relevant interactions are indicated with black dashes. The 2Fo-Fc electron-density map has been contoured at 1.0σ level.</p

MsPrsA quaternary structure assessment.

<p>(A) 10% SDS-PAGE of pure recombinant MsPrsA: molecular weight markers are on the left (M) and the pure enzyme on the right showing a molecular weight of about 37 KDa. (B) Elution profile of MsPrsA on a HiPrep 16/60 Sephacryl 200 High Resolution pre-packed column. In the inset, the arrow indicates the MsPrsA elution volume on the experimentally determined calibration curve.</p

Substrate specificity for different PPi donors and inhibition by purines ribonucleoside diphosphate.

<p>The enzyme activity was determined by using a continuous spectrophotometric coupled assay as described in Materials and Methods. (A) The MsPrsA activity was measured at a concentration of 60μM for R5P, and of 100μM for each PPi donors tested. (B) Inhibition curve for ADP. The MsPrsA activity was measured at a fixed concentration of 60 μM for R5P and 100 μM of ATP, and varying the concentration of ADP (0.2μM-1mM); The data represent the average of three independent experiments.</p

Protein yield from 2 L culture.

<p>Protein yield from 2 L culture.</p

Kinetics parameters of MsPrsA in presence of Pi 50mM.

<p>Kinetics parameters of MsPrsA in presence of Pi 50mM.</p

Modeled <i>M</i>. <i>smegmatis</i> R5P and ADP-Mg²⁺ binding sites superposed on the structure of hPrsA-I; hPrsA-1 and corresponding residues are shown in light blue and MsPrsA and corresponding residues are shown in white.

<p>(A) Ribbon representation of the overall structure of MsPrsA superimposed to hPrsa-I in which the R5P and ADP drawn as ball-and-sticks and represented with the same orientation reported in the respective model of the enzyme-ligand complex. (B) The R5P binding site on the left together with the schematic diagram of MsPrsA residues interacting with the ligand. (C) The ADP-Mg²⁺ binding site on the left together with the schematic diagram of MsPrsA residues interacting with the ligand.</p