PCR primers.

<p>Mutated codons are shown in bold type.</p><p>Restriction endonuclease cleavage sites and 6xHis are written in italics and underlined, respectively.</p

Selected contact between MtQAPRTase and PZA.

<p>Selected contact between MtQAPRTase and PZA.</p

Inhibitory effect of PZA or POA on MtQAPRTase activity and the calculated IC₅₀ values.

<p>(A) Chemical structures of QA, PZA, and POA are shown. Either 1 mM of PZA or POA was incubated with MtQAPRTase at pH 7.2 (B, left) and pH 6.2 (B, right) at 37°C for 30 min. After incubation, reactions were stopped and the products analyzed using HPLC. The IC₅₀ values are indicated in (C). N.D. mean not detected. Data from three separate experiments are represented as mean ± standard error.</p

Kinetic studies of MtQAPRTase.

<p>The Michaelis–Menten plots for enzyme activity were generated in the presence of different concentrations of PRPP (A) and QA (B) as shown in the figure. Reaction mixtures (50 mM KH₂PO₄ [pH 7.2], 6 mM MgCl₂, various concentrations of QA or PRPP, and 1.62 µM of MtQAPRTase) were incubated at 37°C over 20 min. Kinetic studies were performed using reaction mixtures that contained various concentrations of PRPP and a fixed concentration of QA (0.3 mM) as the substrate; conversely, kinetic studies were also performed using reaction mixtures that contained various concentrations of QA and a fixed concentration of the PRPP (1.0 mM) as the substrate. Standard error for three independent experiments is indicated by the bars.</p

Purification of recombinant QAPRTase from <i>M. tuberculosis</i> H37Rv.

<p>Purification of recombinant QAPRTase from <i>M. tuberculosis</i> H37Rv.</p

Schematic representation of the reaction catalyzed by QAPRTase.

<p>Schematic representation of the reaction catalyzed by QAPRTase.</p

Temperature and pH optima. MtQAPRTase was incubated at the various temperatures (A) and at pH 2.2–11.0 (B) for 30 min.

<p>The optimum level of MtQAPRTase activity is denoted with arrows.</p

SDS-PAGE analysis and determination of the molecular mass of recombinant MtQAPRTases.

<p>A) The WT and mutant enzymes are shown above the designated lane. Approximately 5 µg of each protein was loaded on a 5–20% SDS-polyacrylamide gel. Lane M, protein size markers; lane 1, WT MtQAPRTase; lane 2, Arg105′-Ala; lane 3, Arg139Ala; lane 4, Arg162Ala; and lane 5, Lys172Ala. B) Estimation of the molecular mass of recombinant MtQAPRTase using Superdex 200 gel-filtration chromatography. The elution position of MtQAPRTase is indicated by an arrow. Protein standards were as follows: aprotinin (6.5 kDa); ribonuclease A (13.7 kDa); carbonic anhydrase (29.0 kDa); ovalbumin (44.0 kDa); conalbumin (75.0 kDa); aldolase (158.0 kDa), and ferritin (440.0 kDa). <i>K_av</i> values were calculated using the following equation: <i>K_av</i>  =  (<i>V_e−V_o</i>)/(<i>V_c−V_o</i>) is the column void volume, <i>V_e</i> is the elution volume, and <i>V_c</i> is the geometric column volume. The <i>V_o</i> value used was the <i>V_e</i> of Blue Dextran 2000. <i>M_r</i> indicates the molecular weight.</p

Molecular docking studies.

<p>The binding mode of PZA or QA was shown within the catalytic site of MtQAPRTase. The A subunit of MtQAPRTase is depicted in light orange while the B subunit is depicted in pale cyan. The overall structures and the catalytic site on MtQAPRTase are shown on the left and right, respectively. The models of complex structures are indicated as follows: (A) WT MtQAPRTase–PZA and (B) WT MtQAPRTase–merging with PZA [cyan] and QA [green]. The dotted line indicates hydrogen bonding (A and B), and the distance between amino acid residues and PZA/QA is indicated as well.</p

HPLC analysis of substrates and products.

<p>The enzymatic activities of MtQAPRTase were determined using QAPRTase assays with PRPP as the substrate in the absence (A) and presence (B) of purified recombinant MtQAPRTase. NAMN and QA eluted at 6.0 and 12.5 min.</p