G6PDH activity of wild type and mutant enzymes as a function of NAD⁺ or NADP⁺ concentration.

<p>NADP⁺-dependent activity (black circles) and activity with NAD⁺ (white circles) was measured at nearly saturating co-substrate concentrations (bottom right corner). The data was fitted to the Michaelis-Menten equation and the Pearson correlation coefficient is indicated. The values for the kinetic parameters are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152403#pone.0152403.t001" target="_blank">Table 1</a>.</p

G6PDH activity of K18T mutant enzyme as a function of NAD⁺ or NADP⁺ concentration.

<p>NADP⁺-dependent activity (black circles) and activity with NAD⁺ (white circles) was measured at nearly saturating co-substrate concentrations (bottom right corner). The data was fitted to the Michaelis-Menten equation and the Pearson correlation coefficient is indicated. The values for the kinetic parameters are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152403#pone.0152403.t004" target="_blank">Table 4</a>.</p

Kinetic parameters of bacterial G6PDHs.

<p>Kinetic parameters of bacterial G6PDHs.</p

Evolution of the bacterial G6PDHs with regard to cofactor specificity and sequence motifs.

<p>Bayesian tree built from a structurally guided multiple sequence alignment. The species for which kinetic data are available are colored according to their cofactor specificity: red, NADP⁺ specific; orange, NADP⁺-preferring; yellow, dual enzymes. The main clusters are surrounded by a black line and for each one the phylum of the species involved are named. In addition, the sequence logos in terms of probabilities for the sequence motifs aligning with the structural loops containing K18 and R50 (loops β1-α1 and β2-α2) of <i>Ec</i>G6PDH are displayed. The posterior probabilities are shown for each node.</p

Interactions with cofactors at the nucleotide-binding site of the <i>Ec</i>G6PDH model.

<p>Left panel: Time course (sampled every 50 ps; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152403#sec002" target="_blank">Materials and Methods</a> for definition) of hydrogen bonds formed between NAD⁺ (lower) or NADP⁺ (upper) and the binding site residues, during the MD simulation. Interactions with the backbone and side chain atoms were counted separately. Right panel: Free energy contribution of the binding-site residues to cofactor binding, as calculated by MM/PBSA. The transparent surface representation is colored according to an energy scale, going from blue (no interaction) to red (the highest contribution).</p

Double mutant cycle diagram.

<p><i>i</i> and <i>j</i> represent the side chain residues in the wild type enzyme. The replacement by Ala is named “0”, so the wild type enzyme is denoted as (<i>i</i>,<i>j</i>) and the double Ala mutant as (0,0). The free energy change for the (<i>i</i>,<i>j</i>)→(0,<i>j</i>) transition, ΔG_a, corresponds to the lack of the side chain <i>i</i> in the presence of the side chain <i>j</i>. ΔG_b stands for the opposite transition, (<i>i</i>,<i>j</i>)→(<i>i</i>,0). For the lack of the side chain <i>j</i> in absence of <i>i</i>, (0,<i>j</i>)→(0,0), and the lack of the side chain <i>i</i> in absence of <i>j</i>, (<i>i</i>,0)→(0,0), the energies are expressed as ΔG_c and ΔG_d, respectively.</p

Kinetic parameters of the K18T mutant enzyme.

<p>Kinetic parameters of the K18T mutant enzyme.</p

Energetic coupling between K18 and R50 of <i>Ec</i>G6PDH.

<p>The binding free energy changes of the transition state complex with NADP⁺ (A) or NAD⁺ (B) is analyzed as a double mutant cycle. In the case of the NADP⁺ complex, for each residue its contributed energy changes by about 0.6 kcal/mol depending on the presence or absence of the second positively charged residue. In the case of NAD⁺ binding, the coupling between these residues is less significant.</p

Patient baseline characteristics by GFR.

Patient baseline characteristics by GFR.</p

S3 Data -

(XLS)</p