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.
Highlights
Phenazine biosynthesis protein PhzF (E.C. 5.3.3.17) is an example of an enzyme that catalyzes an unusual reaction for which substantially different mechanistic routes can be proposed
Together with a tightly bound water molecule, E45 is involved in the stereospecific tautomerization of AHCDC (4) to AOCHC (5), revealing how PhzF coordinates a water-free with a water-dependent catalytic step and explaining why only a small number of O-alkylated derivatives of dihydro-3-hydroxy anthranilate (DHHA) (3) are accepted as substrates
In order to provide additional evidence for a [1,5]-hydrogen shift in the isomerization of DHHA (3) by PhzF and for the investigation of kinetic isotope effects (KIEs), we developed a synthetic route towards enantiomerically pure (2 S,3 S)-DHHA (3) that allows for deuteration at C3, adapting a synthetic approach by Steel and coworkers14–17
Summary
Phenazine biosynthesis protein PhzF (E.C. 5.3.3.17) is an example of an enzyme that catalyzes an unusual reaction for which substantially different mechanistic routes can be proposed. The phenazines are a class of over 150 bacterial secondary metabolites with redox properties that enable them to act as broad specificity antibiotics as well as virulence and survival factors e.g. in infections caused by the opportunistic pathogen Pseudomonas aeruginosa1, 2 Their biosynthesis involves the condensation of two chorismate-derived precursors into the symmetrical phenazine scaffold (Fig. 1A). The migrating C3-proton of DHHA (3) is expected to be rather non-acidic, suggesting that it cannot be moved by amino acid residues of a cofactor-free enzyme like PhzF Together, these findings suggest that PhzF may not use the essential glutamic acid E45 as a proton shuttle as originally proposed by us, but rather catalyzes a sigmatropic [1,5]-hydrogen shift, i.e. a concerted proton movement that is controlled by frontier orbital symmetry. Together with a tightly bound water molecule, E45 is involved in the stereospecific tautomerization of AHCDC (4) to AOCHC (5), revealing how PhzF coordinates a water-free with a water-dependent catalytic step and explaining why only a small number of O-alkylated derivatives of DHHA (3) are accepted as substrates
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