Investigating the Reaction Mechanism of F₄₂₀-Dependent Glucose-6-phosphate Dehydrogenase from <i>Mycobacterium tuberculosis</i>: Kinetic Analysis of the Wild-Type and Mutant Enzymes

F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using F₄₂₀ cofactor as the hydride transfer acceptor, within mycobacteria. A previous crystal structure of wild-type FGD led to a proposed mechanism suggesting that the active site residues His40, Trp44, and Glu109 could be involved in catalysis. We have characterized the wild-type FGD and five FGD variants (H40A, W44F, W44Y, W44A, and E109Q) by fluorescence binding assays and steady-state and pre-steady-state kinetic experiments. Compared to wild-type FGD, all the variants had lower binding affinities for F₄₂₀, thus suggesting that Trp44, His40, and Glu109 aid in F₄₂₀ binding. While all the variants had decreased catalytic efficiencies, FGD H40A and W44A were the least efficient, having lost ∼1000- and ∼2000-fold activity, respectively. This confirms a crucial catalytic role for His40 in the FGD reaction and suggests that aromaticity at residue 44 aids catalysis. To investigate the proposed roles of Glu109 and His40 in acid–base catalysis, the pH dependence of kinetic parameters has been determined for the E109Q and H40A mutants and compared to those of the wild-type enzyme. The log <i>k</i>_cat–pH profile of wild-type FGD and E109Q revealed two ionizable residues in the enzyme–substrate complex, while H40A displayed only one ionization event. The FGD E109Q variant displayed pH-dependent kinetic cooperativity with respect to the F₄₂₀ cofactor. The multiple-turnover pre-steady-state kinetics were biphasic for wild-type FGD, W44F, W44Y, and E109Q, while the H40A and W44A variants displayed only a single phase because of their reduced catalytic efficiency

The F₄₂₀ production profile from <i>M. smegmatis</i> cells over–expressing the FbiABC construct.

<p>The F₄₂₀–6 and F₄₂₀–7 species constitute the main species as deduced using mass spectrometry.</p

The molecular structures of F₄₂₀, the flavins and NADP⁺.

<p>(A) Schematic representation of F₄₂₀ showing different parts of the molecule, whereas (B) and (C) show the molecular structures of the flavins and NADP⁺, respectively. The atoms involved in oxidoreductive reactions are numbered in all structures.</p

The proposed biosynthetic pathway for cofactor F₄₂₀.

<p>The FbiA, B and C are Mycobacterial proteins whereas the CofA, B, C, D, E, G and H are Archaeal proteins involved in the biosynthetic reactions. The pathway to formation of the activated phospholactate moiety (LPPG) is yet to be experimentally established in Mycobacteria. In some Archaeal species an α–linked terminal glutamate residue caps the γ–linked poly–glutamate tail, the addition of which is catalyzed by CofF.</p

Deazaflavin–dependent reactions in different (micro)organisms.

<p>Deazaflavin–dependent reactions in different (micro)organisms.</p

FO/F₄₂₀ production by <i>M. smegmatis</i> cells expressing different recombinant proteins.

<p>(A) comparative FO/F₄₂₀ production between wild type strain and six different recombinant strains expressing proteins involved in F₄₂₀ biosynthesis or metabolism. (B) FO/F₄₂₀ production using co–expression vector compared to the wild type strain. (C) F₄₂₀ production ratio by <i>M. smegmatis</i> cells expressing FbiABC construct over wild type strains for eight days. (D) Effect of iron/sulphur and L–glutamate/manganese additives on the F₄₂₀ production (FO is shown as black and F₄₂₀ as grey). In panels (A), (B) and (D) the error bars are derived from experiments carried out in triplicate.</p

Functional assay of Rv0132c–Δ38 protein.

<p>The FGD activity was assessed for Rv0132c–Δ38 protein and <i>Mtb</i>–FGD1 as a positive control. <i>Mtb</i>–FGD1 shows a decrease at 420 nm absorbance (green and red lines), whereas Rv0132c–Δ38 protein indicated no change in the absorbance (yellow and blue lines). The same results were observed using various concentrations of Rv0132c–Δ38 protein in the presence of different concentrations of glucose-6-phpsphate. The graph shows assays containing 1 µM of each enzyme, 25 µM F₄₂₀ with 0.1 mM (green and yellow lines) and 1 mM (red and blue lines) glucose-6-phosphate.</p

Structural comparison of Rv0132c with FGD1.

<p>(A) Amino acid sequence alignment. The secondary structure elements for FGD1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045003#pone.0045003-Bashiri2" target="_blank">[5]</a> are shown above the sequence. FGD1 residues that hydrogen bond with F₄₂₀ or the phosphate group of glucose-6-phosphate are indicated below the sequence by F and asterisk, respectively. The twin arginines in the Tat motif and the critical cysteine residue in the lipobox motif are shown in red in the Rv0132c signal sequence. (B) Superposition of the FGD1 (orange) crystal structure on the modeled Rv0132c (cyan). The F₄₂₀ cofactor (green) bound to FGD1 is shown in stick representation. Replacement of helix α₉ with a smaller loop extends the active site cavity in Rv0132c. For details of FGD1 structure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045003#pone.0045003-Bashiri2" target="_blank">[5]</a>.</p

Rv0132c export is Tat dependent.

<p>Equalized whole cell lysates (WCL) from wild type (WT) and <i>ΔtatC M. smegmatis</i> expressing Rv0132c-HA were fractionated to generate cell wall (CW), cytoplasmic membrane (CM), and soluble (SOL) fractions. Fractions were separated by SDS-PAGE and proteins were detected with an anti-HA antibody. Native GroEL was detected as a cytoplasmic control. Rv0132c-HA was exported to the CW and CM fractions in wild type <i>M. smegmatis</i>, but it was not exported in the absence of a functional Tat pathway.</p

Export of an Rv0132cSS–'BlaC¹ fusion protein is dependent on the Tat pathway.

1<p>'BlaC = truncated BlaC lacking its native signal sequence.</p>2<p>All strains were resistant to 20 µg/mL kanamycin due to the vector resistance marker. The presence (+) or absence (−) of carbenicillin resistance was determined by colony growth on LB–agar plates plus 20 µg/mL kanamycin and 50 µg/mL carbenicillin for <i>M. smegmatis</i> and 7AGT plates plus 20 µg/mL kanamycin and 50 µg/mL carbenicillin for <i>M. tuberculosis</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045003#s2" target="_blank">materials and methods</a> for additional experimental details.</p>3<p>Carbenicillin resistance was determined after 4–7 days.</p>4<p>Carbenicillin resistance was determined after 21 days.</p