Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF

Abstract

Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development.

Figure 1

Phenazine biosynthesis and the role of PhzF. (A) Overview over the phenazine biosynthesis pathway. PhzE converts chorismic acid (1) to (5 S,6 S)-6-amino-5-(1-carboxyethenyloxy)-1,3-cyclohexadiene-1-carboxylic acid (ADIC, 2), which is then hydrolyzed to (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilic acid (DHHA, 3) by PhzD. PhzF (red box) catalyzes isomerization to (1 R,6 S)-6-amino-5-oxo-2-cyclohexene-1-carboxylic acid (AOCHC, 5) via (1 R,6 S)-6-amino-5-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (AHCHC, 4) by inducing a [1,5]-hydrogen shift (red H-atom) and a stereospecific tautomerization (green and magenta H-atoms). PhzB then condenses two AOCHC molecules (5) to hexahydrophenazine-1,6-dicarboxylic acid (HHPDC, 6) before oxidation steps involving PhzG generate dihydro-phenazine-1-carboxylic acid (PCAH₂, 7) and dihydro-phenazine-1,6-dicarboxylic acid (PDCH₂, 8) as precursors for strain-specific phenazine derivatives. (B) Crystal structures of the PhzF homodimer in the open (PDB entry 5IWE) and closed (PDB entry 1U1W) conformation. The magnified insert on the left shows O-ethyl-DHHA (20) bound to the active center of the open form, whereas the insert on the right shows the substrate analogue 3OHAA (22) bound to the closed form. The green mesh displays |F_O-F_C| difference electron density at 3 σ and indicates that E45 may be protonated in this complex. This figure was prepared with PyMOL.

Figure 2

Synthesis of C3-deuterated DHHA. Diels-Alder reaction of bromofuran (10) and (E)-nitroacryl ethylester (12) furnishes the racemic bicyclic intermediate rac -13, which is then deuterated by reductive debromination in the presence of DCl. The desired enantiomer 14 is obtained by kinetic resolution with pig liver esterase (PLE). Treatment with potassium hexamethyldisilazide (KHDMS) and deprotection then yields the desired C3-deuterated DHHA (d-3). BOC: tert-butyloxycarbonyl; DCM: dichloromethane; DIPEA: N,N-diisopropylethylamine; 4-DMAP: 4-dimethylaminopyridine; DMF: N,N-dimethylformamide; EDC: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; MsCl: methanesulfonylchloride; NBS: N-bromo succinimide; TFA: trifluoroacetic acid; THF: tetrahydrofuran.

Figure 3

¹H-NMR spectroscopic data. Synthetic DHHA (3, A) and C3-deutero-DHHA (d-3, B) have been incubated with PhzF for the indicated times (C–E). Resonance assignments for DHHA (3) and AOCHC (5) are indicated by colored boxes. The grey rectangle marks enzyme buffer components. Panel F shows an NOE experiment to assign proton resonances at C4 of AOCHC (5). The spectra demonstrate that PhzF recycles the proton at C3 of DHHA (3) to C1 of AOCHC (5; red rectangles) and catalyzes a stereospecific tautomerization with incorporation of a solvent-derived proton at C4 of AOCHC (5; orange and magenta rectangles).

Figure 4

Investigated mechanisms of the isomerization reactions catalyzed by PhzF. (A) Hypothetical mechanisms of the [1,5]-proton shift from DHHA (3) to AHCDC (4). (B) Hypothetical mechanisms for the tautomerization of AHCDC (4) to AOCHC (5). Energy values for the activation energy barriers of the chemical steps shown in the figure (ΔG^≠) have been obtained by QM/MM methods. Together, these values suggest that PhzF initiates isomerization of DHHA (3) by employing E45 to shuttle a proton from C3 to C1 (A, iii), followed by a proton-relay mechanism that stereospecifically tautomerizes AHCDC (4) to AOCHC (5; B, c).

Figure 5

Energy profile of the complete catalytic cycle of PhzF. Calculated structures of intermediates are shown at the top, the most important chemical steps are shown at the bottom of the figure. Letters in circles correspond to respective states of the energy profile.

Figure 6

Determination of enzyme kinetic parameters of PhzF. The reaction velocity vs. substrate concentration for DHHA (3) or C3-deuterated DHHA (d-3) in H₂O at pH 7.5 is shown. Enzyme kinetic parameters for DHHA (3) were determined as v_max = 100.34 ± 5.80 nmol s⁻¹ mg⁻¹ (k_cat = 3.23 ± 0.19 s⁻¹) and K_M = 517 ± 57 µM, or as v_max = 10.60 ± 0.27 nmol s⁻¹ mg⁻¹ (k_cat = 0.34 ± 0.01 s⁻¹) and K_M = 311 ± 14 µM for C3-deuterated DHHA (d-3), respectively.

Figure 7

Substrate preferences of PhzF. (A) Turnover of isosters of DHHA, (B) conversion of O-alkylated DHHA-derivatives. Colors indicate velocities with which the respective compound is isomerized (green: fast; yellow: slow; red: no turnover).